All the newly synthesized compounds were confirmed using various physical, spectroscopic, and analytical data. The in vitro anticancer activities of each compound were evaluated against colorectal carcinoma Colon cancer (HCT-116) and Epdermoid carcinoma (HEP2) and also Human lung fibroblast normal cell line (WI38) compared with Doxorubicin. The results showed that Compounds 2a, 4b and 4d exhibited more potent inhibitory activity for Epdermoid Carcinoma (HEP2) compared with doxorubicin. For colon carcinoma cells (HCT-116) Compounds 2a, 2d and 4b gave the strongest activity among all compounds compared with doxorubicin. Moreover, all designed structures were docked into the active site of VEGFR2 and FGFR1 proteins. The result reveals that compound 2b and have the strongest inhibitory activity of the VEGFR2 and FGFR1 proteins indicating that these substances might conceivably operate as VEGFR2 and FGFR1 inhibitors and hence might take role in anticancer activities with various binding interactions. The 3D-QSAR models produced strong statistical results since they were defined by PLS factors 4 and confirmed by parameters as R2, R2 CV, Stability, F-value, P-value, RMSE, Q2, and Pearson-r.The present study involves synthesis a new series of α-aminophosphonates  Heterocyclic compounds play a vitally important role in medicinal chemistry and are receiving healthy funding with a wider range of potential for processing and synthesizing a wide variety of pharmacological characteristics. Along with being widely present in natural products2. There are numerous therapeutic applications for heterocyclic compounds, including pharmacological and medicinal properties13. Pyrazole-containing compounds represent one of the most significant families of N-heterocycles. Due to their shown utility and adaptability as synthetic intermediates in the synthesis of key molecules in biological, physical–chemical, material science, and industrial applications, As a result, it is extremely desirable to synthesize pyrazole derivatives with a variety of structural characteristics, and numerous researchers are still working to create this useful scaffold and discover new and better uses for it. Moreover, The pyrazole moiety has been shown to have pharmacological potential by its presence in a variety of therapeutic agents, including the antipsychotic CDPPB, the anti-obesity medication rimonabant, the analgesic difenamizole, the H2-receptor agonist betazole, and the antidepressant fezolamide  has become an attractive validated therapeutic target notably in cancer diseases.According to literature survey, a series of some new pyrazolyl derivatives were synthesized and investigated for their antitumor activity against different cancer cell lines. These results indicated that the pyrazolyl derivatives displayed antiproliferative activity in a range of human tumor cell lines, including HCT116 human colon carcinoma and Epdermoid Carcinoma (HEP2), Epdermoid Carcinoma (HEP2) and also Human lung fibroblast normal cell line (WI38) besides different tumor cell lines. For the moment, researchers have been drawn to the design of more potent pyrazole derivatives having great diversity of biological activity  are most important group of organophosphorus compounds that are structural analogues of amino acids where a carboxylic substituent is substituted by phosphonic acid or related groups20. Recently, α-aminophosphonates have received enormous attention from scientific researchers in pharmaceutical and medicinal chemistry due to they have extremely high levels of wide biological activities such as anti-HIV, peptidemimics, antibacterial, antibiotics, inhibitors of serine hydrolase, herbicidal, antiviral, anticancer, enzyme inhibitors, antifungal, and anti-proliferative, enzyme inhibitors, inhibitors of serine hydrolase anti-Alzheimer and apoptosis inducing22. Moreover, α-aminophosphonate derivatives containing a pyrazole moiety showed a significant inhibitory effect on acetylcholinesterase (AChE)23. Vascular endothelial growth factor (VEGF) is a key signalling molecule that controls the tumor angiogenesis process. VEGF overexpression was discovered in a number of cancer, including Epdermoid carcinoma (HEP2) and Colon cancer (HCT-116). All VEGF responses in endothelial cells are mediated by VEGFR-2. Therefore, VEGFR-2 should be the primary target of any new drugs being developed to treat cancers in humans that are dependent on angiogenesis24.The biological and therapeutic properties of 5-amino-pyrazoles as well as their flexibility in synthetic processes have recently drawn a lot of attention and given them a prominent position25.Various methods for the synthesis of α-aminophosphonates were reported. However, one pot Kabachnik-Fields reaction, a one-pot multicomponent synthesis involving an amine, an aldehyde, and a phosphite in the presence of a Lewis acid catalyst remains the most efficient, simple, general, and high yielding method28, we have designed and synthesized a series of new α-aminophosphonates derivatives bearing pyrazole moiety and were evaluated against references and cancer cell lines. Additionally, the newly synthesized compounds’ biological activity was examined in connection to changes in their molecular and electronic structure using density functional theory in an effort to connect theoretical and experimental results. To determine the target enzyme and the most active compound's mechanisms of action, a molecular docking simulation will be run.Based on these facts and keeping in view the wide range of biological activities of pyrazole moieties, and aminophosphonate scaffolds, in this work, we expect that the incorporation of these moieties in the same scaffold structure may lead to good activities and potent powerful anticancer medicines. Thus, as a continuation of our prior work in the synthesis of biologically active heterocycles1/or 3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-5-amine 2, different aromatic aldehydes, triethylphosphite/or triphenylphosphite in CH2Cl2 in the presence of lithum perchlorate as a Lewis acid catalyst via one-pot Kabachnik–Fields25, gives Diethyl{(3-(Aryl)-1-phenyl-1H-pyrazol-5-ylamino)(Aryl′)} methyl phosphonate 2a-f and Diphenyl{(1-(Aryl)-3-phenyl-1H-pyrazol-5-ylamino)(Aryl′)} methyl phosphonate 4a-d, respectively, in a very good yield  appeared at 1104–1011 cm–1 and υ (P–CH) is absorbed at 765–701 cm–1.The CH aromatic stretching band is absorbed at 3162–3025 cm–1, while the CH aliphatic bands are appeared at 2982–2853 cm–1. Finally, NH/OH groups are absorbed at 3464–3413 cm–1.A new series of α-aminophosphonates bearing pyrazole skeleton were synthesized via three component Kabachnic-Fields reaction of 3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-5-amine 1H-NMR (DMSO) spectra of α-aminophosphonate derivatives, 2a-f, showed the following signals: δ 5.02–5.76 as singlet was assigned to P–CH and the singlet signals at the range of δ 6.23–7.12 were attributed to pyrazole–CH. The Ar–H signals appeared at the range of δ 6.32–9.26 ppm, also the –NH proton appeared at δ 5.61–5.99 and signals at δ 6.42–7.21 were attributed to pyrazole–CH. The methylene and methyl protons of P–O–CH2CH3 resonated as quarter and triplet respectively at δ 3.62–4.39 and 1.26–1.39 ppm. The 13C-NMR (DMSO) of the studied compounds are shown as the following signals at δ 146.42–167.94 and 143.32–158.29 (C=Nimine) and (C=N pyrazole ring), respectively, 139.33–148.97 (C–NO2), 54.04–66.82 , 81.93–89.39 (CHpyrazole), 114.35–148.34 (CAromatic). Additionally, a peak at m/z corresponding to the product’s molecular ion was visible in the mass spectrum for the all product. By using elemental microanalysis to characterize the examined substances, it was found that the calculated value and the measured value agreed well. The selected spectroscopic data are reported in the (“).The  in the “” sectionNitrogen-containing heterocycles exhibit anticancer effect in various types of cancer through inhibiting cell growth and induction of cell differentiation and apoptosis.29.Moreover, therapeutic drugs that inhibiting EGFR and VEGFR-2 can enhance the effectiveness of cancer therapy and resolve resistance issues30.The cytotoxic potency of the synthesized phosphonates, 2a-f and 4a-d was determined in vitro using the standard MTT method31 against Colorectal carcinoma Colon cancer (HCT-116) and Epdermoid Carcinoma (HEP2) and also Human lung fibroblast normal cell line (WI38), using Doxorubicin as a positive control. The IC50 values were estimated for each compound and the results are shown in , while the rest compounds have moderate to weak activity against the same cell line as shown in Table HCT-116) Compounds 2a, 2d and 4b gave the strongest activity among all compounds. Finally, all the studied phosphonate compounds have moderate to weak cytotoxicity activity on the normal cell WI-38.To create novel molecules with high inhibitory potentials, pyrazole derivatives were widely utilizeder HCT-11 and Epde2a which contain 2, 3-diOH electron donating substituent and compounds 2d and 4b which have electron withdrawing NO2 group, are the most potent derivatives against the tested cancer cell lines and also have low cytotoxic activity on the normal cell line The variety of substituents in the aryl aldehyde moiety of AAPs is important for the wide range of cytotoxic activity against different cell lines , Figs. , 5 and 6(ii)2b which has one OH group in position 3 in the aryl aldehyde moiety is in more active compound towards the cell lines, but compound 2a which has 2-OH groups in position 2 and 3 in the aryl aldehyde moiety enhanced the cytotoxic activity and showed a medium cytotoxic activity against the normal lung cell WI-38 compared with doxorubicin which has OH moieties.Compound (iii)2c which has one Cl group in position 3 in the aryl aldehyde moiety enhance the biological activity against the tested cell lines.Compound (iv)2d, and the di-phenyl phosphonate compound 4b which have electron withdrawing substituent NO2 in the aryl aldehyde moiety have potent cytotoxic activity against the cell line among all compounds.The di-ethyl phosphonate, (v)4d which has electron donating group N(CH3) in the aryl substituted aldehyde moiety is more active than the di-ethyl phosphonate of the same substituent 2f which is may be due to the increasing number of phenyl rings which increase the resonance and accordingly increase the antitumor activity.The di-phenyl phosphonate compound (vi)2a improves potency more than compound 2d which have an electron withdrawing group NO2 . Furthermore, most reactions are caused by the interaction of one moiety's HOMO and another’s LUMO.The “HOMO energy level” that occupies the highest molecular orbital is essentially an electron donor molecular orbital. The lowest unoccupied molecular orbital, or “LUMO”, is mostly used as an electron acceptor. Both orbitals were called frontier molecular orbitals (FMOs) , and chemical descriptors for the examined compounds. These descriptors can be assessed using the formulae provided by Pearson, and Padmanabhan et al.34.Table 34. and is equal to the maximum number of electrons moved in the chemical process (ΔNmax).The additional electronic charge is derived by the formula provided by Geerlings et al. and Pearson2b > 2a > 2d > 2f > 4a > 2e > 2c > 4b > 4c > 4d. This order indicates that the stability of 4d is higher than that of other compounds.The gas phase energy decreases with the order: EHOMO and ELUMO values are both negative, suggesting that the isolated compounds are stable36.The 4a (− 3.835 eV) > 2a (− 3.835 eV) > 2b (− 3.925 eV) > 2c (− 4.021 eV) > 2e (− 4.162 eV) > 4d (− 4.167 eV) > 2f (− 4.214 eV) > 2d (– 4.378 eV) > 4b (− 4.445 eV) > 4c (− 4.482 eV).Hard molecules have larger HOMO–LUMO disparities, whereas soft and active molecules have smaller energy differences. The chemical potential ‘μ’ which quantifies electrons’ ability to escape from the equilibrium structure, is decreased as follows: max is the reaction index, which gauges the bond energy’s stability. The computation of ΔNmax reveals that 2f has the greatest value (3.8900 e), which is greater than other compounds, showing its high electron acceptance.ΔN4a is greater than other compounds’ values which may increase its hydrophilic nature and, as a result, its biological potency.The dipole moment value of the Using the computation findings provided in Table 2 was obtained, where R2 is a statistical number indicating how closely the regression line approximates the true data points. As a result, a good connection was discovered for the examined chemicals, as shown in  and average local ionization energy (r) of the investigated compounds have been demonstrated to be dynamic guides to its reactive performance37.Electrostatic potential V,maxS and the most negative V,minS.The most positive S, with its positive averages The whole surface potential value S,max and most negative ,S,min and the average over the surface of the local ionization energy .S,aveMost positive Internal charge transfer  Π, is derived as a sign of internal charge separation and is present even in molecules with zero dipole moment due to symmetry.38.The variances,An electrostatic balancing parameter ν, that indicates the degree of equilibrium between positive and negative potentials; it has a maximum value of 0.25 when Figure 2a has the largest Π with value 12.52 kcal/mol followed by 2b. This is because to their strong polar structures. While 2c exhibits lowest value of Π = 10.16 kcal/mol and may be related to its structural symmetry.Table 2a are strong with balanced with ν = 0.250. While, the weaker surface potentials with ν = 0.194 is related to 2e. Furthermore, the largest value of 2a shows the strong and variable + ve and -ve surface potentials.The positive surface potential (VS(r) and S(r) on surfaces of 2a as a representative example and shows the locations of the various most positive, V,maxS and most negative, V,minS as well as the highest, S,max and lowest, S,min. On a particular molecular surface, there are frequently many local minima and maxima of each attribute. The highest negative electrostatic potential on the surface of 2a is related to the oxygen (O27), VS,min = − 39.1 kcal/mol, followed by a weaker value of − 39.07 kcal/mol on the oxygen (O28). As a result, VS(r) incorrectly guess electrophilic attack to arise specially at the oxygen atom. However, the lowest value of S(r) is found on the (C20), with S,min = 189.27 kcal/mol; as well as, there is an S,min by the nitrogen (N31), but it is significantly higher, 198.28 kcal mol–1. Thus, S(r) demonstrates that the least-tightly-bound electrons, and most reactive are at (C20), correctly suggesting that these sites are more vulnerable to electrophiles. On contrast, the extremely significantly positive electrostatic potential of hydrogen (H50), VS,max = 61.78 kcal/mol, and the VS,min = 7.16 kcal/mol of the hydrogen (H41) show their proclivity for noncovalent H-bonding as a donor.Figure 2b and 2a have the strongest inhibitory activity of the VEGFR2 and FGFR1 proteins, respectively. The sequence of inhibitory activity to the VEGFR2 protein is 2b > 2f > 4a > 2a > 4b > 4d > 2e > 2c > 2d. While the sequence of inhibitory action to the FGFR1 protein is 2a > 2b > 2f > 4d > 4a. Furthermore, 4c doesn’t exhibit any interactions with the VEGFR‐2 protein. Also compounds 4c, 2e, 2d, 4b, and 2c don’t show any interactions with the FGFR1 protein. From these data, one can notice that compound 4c has no interaction with FGFR1 protein and agree with experimental data of in vitro cytotoxicity against WI38 (IC50 > 100). The examined substances demonstrate that H-donor and H-acceptor interactions are the most common kind of interaction with VEGFR2 and FGFR1 receptors  group, PTR1052 to (N=O) and LYS1053 to (N=O) with distances 1.97, 2.22 and 2.74 Å, respectively. While the interaction of 2a with FGFR1 protein  and the RMSD of (0.618 and 0.792 Å), respectively).The re-docking was done to test the docking strategy and efficiency. The re-dockin approach used the same procedures as earlier. The peptide original inhibitor was precisely attached to the active site pocket of the VEGFR2 receptor by completely 5- hydrogen bonds were formed with distances , 2.08, 2.46, and 2.23 Å. While the original inhibitor linked to the active site pocket of FGFR1 receptor through four hydrogen bonds with distances 2.34, 2.17, 2.34, and 2.92 Å as well as π-cation interaction between the aromatic ring and LYS514 with distance 5.51 Å. The re-docked original ligand was overlaid onto the native co-crystallized Figs.  and 13. 40 to assess the models’ prediction abilities 2 substituent is equally good for inhibiting both models. But the electron-withdrawing effect of (NO2) substituents shows good and poor inhibiting. Finally, the (Cl) substituent doesn’t exhibit any effect.The electron-drawing effect is likewise insufficient for distinguishing the inhibitory characteristics of VEGFR2 and FGFR1. Also, the electron-withdrawing effect of N(CH2) ion made compounds are not favorable for VEGFR2 inhibition. On the other hand, the positive ionic effect of (NO2) toward FGFR1 inhibition is good inhibitor except for the compound 2f and 4d. As a result, the compound’s positive ionic action on both inhibitory systems is very instructive in distinguishing between VEGFR2 and FGFR1 inhibition.The positive ionic effect of  and Epdermoid carcinoma (HEP2) and also Human lung fibroblast normal cell line (WI38). From the data, we could conclude that all these novel α-aminophosphonates could inhibit tumor cell lines (HCT-116 and HEP2) below 10 µM by the MTT assay. Moreover, Compounds 2a, 4b and 4d exhibited more potent inhibitory activity for Epdermoid Carcinoma , respectively, compared with doxorubicin (0.6 ± 8.54 μM), while the rest compounds have moderate to weak activity against the same cell line. For colon carcinoma cells (HCT-116) Compounds 2a, 2d and 4b gave the strongest activity among all compounds with IC50 values , respectively, compared with doxorubicin (0.3 ± 5.32 μM). Finally, all the studied phosphonate compounds have good to moderate cytotoxicity activity on the normal cell WI-38. The geometry optimization results using DFT exhibits that the stability of 4d is higher than that of other compounds as well as 2f has the highest electron acceptance while dipole moment of 4a is greater than other compounds’ values which may increase hydrophilic nature of 4a. From molecular docking interaction, the results reveals that compounds 2a shows good interactions with VEGFR‐2 and FGFR1 proteins respectively, this behavior may be due to the highest positive and negative surface potentials of 2a obtained from electrostatic potential calculations. On the other hand, compound 4c doesn’t exhibit any interactions with the VEGFR‐2 protein. Also compounds 4c, 2e, 2d, 4b, and 2c don’t show any interactions with the FGFR1 protein. From these data, one can notice that compound 4c has no interaction with FGFR1 protein and agree with experimental data of in vitro cytotoxicity against WI38 with IC50 values (> 100 μM).In conclusion, this study is based on synthesized and evaluated new series of α-aminophosphonates 41 were synthesized.All data of chemicals and instruments are available in the supplementary file (Sect. S1). In accordance with literature procedures, the compounds aryl substituted pyrazolamines1/or 3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-5-amine 2 (0.01 mol) and appropriate aldehyde derivatives (0.0012 mol) in dry dichloromethane CH2Cl2 (5 ml), triphenyl phosphite and/or triethyl phosphite (0.001 mol) and anhydrous lithium perchlorate LiClO4 (100 mol %) were added. The reaction mixture was stirred at room temperature (48 h) until the completion of the reaction as indicated by TLC. Then CH2Cl2 was evaporated and the α-amino phosphonates were precipitated using methanol. The precipitate was filtered off affording new α-amino phosphonates in good yield.To a stirred solution of 3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-5-amine –1: 3248 (NH), 3029 (CH-Arom.), 2925 (CH-Aliph), 1254 (P=O), 1086 (P–O–C), 761 (P–CH). 1H NMR  δ 1.32 , 3.65 , 5.07 , 6.94 , 7.14–8.00 , 5.70 , 9.41, 9.31. 13C NMR  δ 12.23 (2CH3), 52.47 (2CH2), 54.04 (CH-P), 83.24 (CH-Pyrazole), 152.28 (C=N pyrazole), 148.63 (C–NH), 148.47 (C–NO2), 161.43(C–OH), 122.16–148.34 (Ar–C). MS : m/z = 538.73 [M]+. Anal. Calcd. For C26H27N4O7P (538.16) C, 57.99; H, 5.05; N, 10.40. Found; C, 57.91; H, 4.98; N, 10.34.Isolated yield = 85%, Melting point: 277–279 °C, IR: ν/cm–1: 3413 (NH), 3025 (CH–Arom.), 2917 (CH–Aliph.), 1265 (P=O); 1006 (P–O–C), 700 (P–CH). 1H NMR  δ 1.23 , 4.25 , 5.29 , 6.55 , 7.14–8.72 , 5.71 , 9.06 . 13C NMR  δ 12.23 (2CH3), 59.59 (2CH2), 66.81 (CH-P), 81.93 (CH-Pyrazole), 167.94 (C=N pyrazole), 146.24 (C-NH), 145.54 (C–NO2), 117.44–144.20 (Ar–C). MS : m/z = 522.66 [M]+. Anal. Calcd. For C26H27N4O6P (522.17) C, 59.77; H, 5.21; N, 10.72. Found; C, 59.69; H, 5.16; N, 10.65.Isolated yield = 77%, Melting point: 252–254 °C. IR: ν/cm–1: 3435 (NH), 3029 (CH–Arom.), 2982 (CH–Aliph.), 1193 (P=O), 1104 (P–O–C), 758 (P–CH). 1H NMR  δ 1.26 , 3.62 , 5.35 , 6.23 , 6.72–8.52 , 5.61 . 13C NMR  δ 22.06 (2CH3), 64.01 (2CH2), 66.82 (CH-P), 85.49 (CH-Pyrazole), 146.42 (C=N pyrazole), 143.77 (C–NH), 139.33 (C-NO2), 119.72–137.88 (Ar–C). MS : m/z = 540.13 [M] +. Anal. Calcd. For C26H26ClN4O5P (540.12) C, 57.73; H, 4.84; N, 10.36. Found; C, 57.67; H, 4.79; N, 10.29.Isolated yield = 84%, Melting point: 150–152 °C. IR: ν/cm-1: 3430 (NH), 3068 (CH–Arom.), 2853 (CH–Aliph.), 1230 (P=O); 1083 (P–O–C), 764 (P–CH). 1H NMR  δ 1.39 , 4.32 , 5.52 , 6.81 , 7.13–8.35 , 5.85 . 13C NMR  δ 15.21 (2CH3), 58.29 (2CH2), 59.05 (CH–P), 89.37 (CH–Pyrazole), 148.97 (C = N pyrazole), 144.32 (C–NH), 142.92 (C–NO2), 116.39–135.83 (Ar–C). MS : m/z = 549.12 [M+-2]. Anal. Calcd. For C26H26N5O7P (551.16) C, 56.62; H, 4.75; N, 20.31. Found; C, 56.54; H, 4.69; N, 20.24.Isolated yield = 75%, Melting point: 234–235 °C. IR: ν/cm–1: 3447 (NH), 3162 (CH–Arom.), 2922 (CH–Aliph.), 1239 (P=O), 1069 (P–O–C), 765 (P–CH). 1H NMR  δ 1.31 , 4.39 , 5.49 , 6.42 , 6.72–8.95 , 5.92 . 13C NMR  δ 16.97 (2CH3), 41.90 (2CH2), 66.82 (CH-P), 82.89 (CH-Pyrazole), 151.35 (C=N pyrazole), 150.40 (C–NH), 146.87 (C–NO2), 116.97–139.73 (Ar–C). MS : m/z = 540.80 [M] +. Anal. Calcd. For C26H26ClN4O5P (540.13) C, 57.73; H, 4.84; N, 10.36. Found; C, 57.65; H, 4.77; N, 10.29.Isolated yield = 77%, Melting point: 298–300 °C. IR: ν/cm–1: 3422 (NH), 3065 (CH–Arom.), 2919 (CH–Aliph.), 1267 (P=O); 1011 (P–O–C), 701(P–CH). 1H NMR  δ 2.61 , 1.41 , 3.93 , 5.31 , 6.94 , 7.14–9.41 , 5.99 . 13C NMR  δ 20.61(2CH3), 12.63 (2CH3), 55.16 (2CH2), 64.82 (CH-P), 83.86 (CH-Pyrazole), 161.43 (C = N pyrazole), 152.28 (C–NH), 148.62, 148.47 (2 C–NO2), 119.26–148.34 (Ar–C). MS : m/z = 594.67 [M] +. Anal. Calcd. For C28H31N6O7P (594.20) C, 56.56; H, 5.26; N, 14.13. Found; C, 56.49; H, 5.21; N, 14.07.Isolated yield = 82%, Melting point: 244–246 °C. IR: ν/cm–1: 3432 (NH), 3032 (CH–Arom.), 1234 (P=O), 1015 (P–O–C), 701 (P–CH). 1H NMR  δ 5.34 , 6.84 , 6.32–8.64 , 5.74 , 6.34 . 13C NMR  δ 68.35 (CH–P), 89.37 (CH-Pyrazole), 159.05 (C=N) pyrazole, 158.29 (C–NH), 148.97 (C–NO2), 116.39–135.83 (Ar–C). MS : m/z = 617.04 [M+-1]. found,.Anal. Calcd. For C34H27N4O6P (618.17) C, 66.02; H, 4.40; N, 9.06. Found; C, 65.96; H, 4.32; N, 8.99.Isolated yield = 81%, Melting point: 266–268 °C. IR: ν/cm–1: 3432 (NH), 3065 (CH–Arom.), 2925 (CH–Aliph.), 1235 (P=O), 1025 (P–O–C), 765 (P–CH). 1H NMR  δ 5.49 , 6.61 , 6.75–8.45 , 5.94 . 13C NMR  δ 64.01 (CH–P), 83.77 (CH–Pyrazole), 146.42 (C=N pyrazole), 143.77 (C–NH), 139.33 (C–NO2), 119.72–137.88 (Ar–C). MS : m/z = 647.25 [M] +. Anal. Calcd. For C34H26N5O7P (647.16) C, 63.06; H, 4.05; N, 10.81. Found; C, 62.98; H, 3.97; N, 10.74.Isolated yield = 83%, Melting point: 167–168 °C. IR: ν/cm–1: 3434 (NH), 3063 (CH–Arom.), 2924 (CH–Aliph.), 1237 (P=O), 1064 (P–O–C), 764 (P–CH). 1H NMR  δ 5.29 , 7.12 , 7.31–8.73 , 5.75 , 6.31 . 13C NMR  δ 58.28 (CH-P), 89.39 (CH–Pyrazole), 158.28 (C=N pyrazole), 148.96 (C–NH), 147.26 (C–NO2), 160.41(C–OH), 114.75–140.75 (Ar–C). MS : m/z = 663.91 [M] +. Anal. Calcd. For C34H26N5O8P (663.15) C, 61.54; H, 3.95; N, 10.55. Found; C, 61.49; H, 3.89; N, 10.48.Isolated yield = 79%, Melting point: 198–200 °C. IR: ν/cm–1: 3464 (NH), 3025 (CH–Arom.), 2921 (CH–Aliph.), 1252 (P=O), 1080 (P–O–C), 732 (P–CH). 1H NMR  δ 2.51 , 5.76 , 6.87 , 7.38–9.26 , 5.92 . 13C NMR  δ 20.61 (2CH3), 65.16 (CH–P), 88.98 (CH–Pyrazole), 158.36 (C=N pyrazole), 156.46 (C–NH), 148.27, 146.46 (2 C–NO2), 144.35–143.74 (Ar–C). MS : m/z = 690.81 [M] +. Anal. Calcd. For C36H31N6O7P (690.20) C, 62.61; H, 4.52; N, 12.17. Found; C, 62.55; H, 4.46; N, 12.10.Isolated yield = 80%, Melting point: 212–214 °C. IR: ν/cm31. The human tumor cell lines that used to evaluate the activity are Human lung fibroblast (WI38), Colorectal carcinoma Colon cancer (HCT-116) and Epdermoid Carcinoma (HEP2). The MTT assay is a standard colorimetric assay that is used to measure cell growth. It is used to determine cytotoxicity of potential medicinal agents and other toxic materials. Detailed procedures for the MTT assay method is presented in the supporting information (Sect. S2).The in vitro antitumor activity of the compounds under study in this research was investigated cell growth using the MTT assay41 in Schrödinger’s suite42 was used to optimise the geometry of investigated compounds. The density functional theory (DFT) was utilised to simulate chemical processes and predict material characteristics using the hybrid density functional technique B3LYP-d343 combination with a 6-31G** basis set for examined substances.The Jaguar 11.2 software44.A molecular modeling technique called molecular docking is often used to examine how receptors and investigated compounds interact in detail46. Furthermore, Fibroblast growth factor receptors (FGFR1) amplification is commonly observed in 9–20% of squamous non-small cell lung cancer48. Therefore, the three-dimensional complex structures of HCT-116 (PDB ID: 1YWN) and W-I38 (PDB ID: 5UR1) were retrieved from the Protein Data Bank50 which they are related to the biological targets VEGFR‐2 and FGFR1, respectively. The protein structures were created with the Schrödinger suite’s protein preparation wizard52 which eliminated water molecules (> 5A radius) and small molecules from the structural section, built disulfide connections, and added hydrogens to the PDB structures. Constrained minimization with default parameters was performed on the structure using the optimized potentials for liquid simulations (OPLS-2005) force field. The structures created were used to generate receptor grids for docking.Vascular endothelial growth factor receptor 2 (VEGFR‐2) was found to be crucial for cell survival, which regulates endothelial differentiation in human colorectal carcinoma (HCT‐116)53 in Schrödinger’s suite. The glide program52 in Schrödinger’s suite was used for docking studies. The glide dock XP procedure was used to dock all investigated compounds to the target protein. The G-score value is a letter grade that indicates how well a compound binds to a receptor. The RMSD may also be utilised to compare a binding conformation to a reference binding configuration. The G-score and RMSD values can be identified as potential inhibitors54.The standard drug Doxorubicin and the optimized target compounds using a basis set 6-31G** were intended to be used for docking and were organized using the default methodology of the Ligprep softwareThe docking procedure was confirmed by removing and redocking 1-{4-pyrimidin-5-yl]phenyl}-3-[2-fluoro-5-(trifluoromethyl)phenyl]urea inhibitor from the VEGFR‐2 receptor (PDB ID: 1YWN) and 3--1-{1-[4-(dimethylamino)but-2-enoyl]piperidin-4-yl}-7-(phenylamino)-3,4-dihydropyrimidopyrimidin-2(1H)-one (PDB ID: 5UR1) inhibitor from the FGFR1 receptor into the active sites using Schrödinger’s suite software using the same approach including function parameters were unaltered in the process. This was done to ensure that the inhibitor binds to the active site pocket accurately and with less variance than the true co-crystallized complex.55. Schrodinger software’s phase module was used to create atom-based QSAR models for the two sets. The dataset should be extremely well aligned with each other56 to derive a statistically valid 3D QSAR model.Three-dimensional QSAR analysis is a high-dimensional QSAR approach in which the descriptors are three-dimensional57 The PLS factor establishes the relationship between the dependent and independent variables. The same collection of training and test set molecules is used for QSAR model creation of VEGFR2 and FGFR1 receptors inhibition. The models’ dependability and utility were validated by utilizing an external collection of 10 active medicinal compounds retrieved from the ChEMBL drug. (https://www.ebi.ac.uk/chembl/).The training set for model creation was chosen at random, with 70% of the molecules in the training set and 30% in the test set for developing models for VEGFR2 and FGFR1 receptors. For regression analysis, the partial least squares regression approach with a PLS factor of four was utilizedSupplementary Information.


In the title compound, both the host and guest are completed by crystallographic twofold symmetry (one carbon atom of the host lies on the rotation axis). The pentagonal-shaped macrocycle has a pair of buteneoxy substituents on one of its faces and one molecule of 1,4-dibromobutane is encapsulated within the cavity of the pillararene, forming a 1:1 inclusion complex. 51H58O10·C4H8Br2, both the host and guest are completed by crystallographic twofold symmetry (one carbon atom of the host lies on the rotation axis). The pentagonal-shaped macrocycle has a pair of buteneoxy substituents on one of its faces and one molecule of 1,4-dibromobutane is encapsulated within the cavity of the pillararene, forming a 1:1 inclusion complex. The terminal alkene parts, which project outwards from the pillararene ring, exhibit positional disorder over two sets of sites in a 0.52 (2): 0.48 (2) ratio. The host and guest interact via C—H⋯O, C—H⋯Br and C—H⋯π interactions and adjacent host molecules interact via C—H⋯O and C—H⋯π bonds.In the title compound, C Single crystals of this pillararene were grown from a solution containing dibromobutane and its structural as well as supramolecular features are discussed.In the present work, an inclusion system comprising butenoxy-substituted pillararene and dibromobutane is reported. The parent pillararene-1-[1–4-di(but-3-en-1-yloxy)]-2,3,4,5-pillar[5]arene [C2/c. The asymmetric unit contains half of the pillararene molecule 2·ButBr2 shows the that 1,4-dibromobutane guest species is threaded inside the pillararene cavity, forming a 1:1 inclusion complex. All of the H atoms of the guest molecule are capable of engaging in non-bonding interactions with pillararene ring, either via C—H⋯O or C—H⋯π interactions. In addition, the pillararene macrocycle is able to connect with the bromine atoms of the dibromobutane by C—H⋯Br interactions. The nature of these various non-bonding interactions are depicted in Fig. 3The crystal structure of Pil(Butenoxy)2·ButBr2 species exhibit intermolecular non-bonding C—H⋯O or C—H⋯π interactions in their crystal network. The multiple non-bonding  interactions  between neighboring Pil(Butenoxy)2.ButBr2 systems are shown in Fig. 4Pil(Butenoxy)2·ButBr2 complex is depicted in Fig. 5a-axis direction.The Pil(Butenoxy)2: Paraformaldehyde  was added to a solution of 1,4-dimethoxybenzene  and 1,4-bis(but-3-en-1-yloxy)benzene  in 1,2-dichloroethane (60 ml) under a nitrogen atmosphere. Boron trifluoride diethyl etherate  was then added to the solution and the mixture was stirred at 0°C for 1 h. MeOH (200 ml) was poured into the mixture to quench the reaction and the reaction mixture was filtered. The residue was dissolved in chloroform (50 mL) and filtered. The filtrate was concentrated to a small volume and adsorbed on silica and column chromatography was performed using a dichloromethane:hexane mixture (60:40 v/v). The second last fraction was the intended pillarene. Yield: 228 mg (16%). 1H NMR  δ: 2.50 , 3.68  3.80 , 3.91 , 5.08 , 5.92 , 6.79 . 13C NMR , δ: 29.8, 29.8, 29.9, 34.4, 56.0, 56.0, 56.0, 56.1, 68.0, 114.3, 114.3, 114.4, 114.4, 115.4, 116.9, 128.3, 128.4, 128.5, 128.6, 128.6, 135.2, 150.1, 151.0, 151.0, 151.0.Synthesis of vinyl-substituted pillararene Pil(Butenoxy)2·ButBr2 inclusion complex: Pil(Butenoxy)2 (20 mg) was dissolved in a solution of dichloromethane and 1,4 dibromo butane . Single crystals of pillararene encapsulated with the dibromobutane guest were grown by slow solvent evaporation after storing the solution in an NMR tube that was kept cold. Crystals suitable for X-ray diffraction were grown in 5 days.Crystal growth of Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314623005886/xu4051sup1.cifCrystal structure: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623005886/xu4051Isup3.cdxSupporting information file. DOI: 10.1107/S2414314623005886/xu4051Isup4.hklStructure factors: contains datablock(s) I. DOI: 2254104CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Integrins have been suggested to be involved in SARS-CoV-2 infection, but the underlying mechanisms remain largely unclear. This study aimed to investigate how integrins facilitate the ACE2-mediated cellular entry of SARS-CoV-2. We first tested the susceptibility of a panel of human cell lines to SARS-CoV-2 infection using the spike protein pseudotyped virus assay and examined the expression levels of integrins in these cell lines by qPCR, western blot and flow cytometry. We found that integrin αvβ1 was highly enriched in the SARS-CoV-2 susceptible cell lines. Additional studies demonstrated that RGD (403–405)→AAA mutant was defective in binding to integrin αvβ1 compared to its wild type counterpart, and anti-αvβ1 integrin antibodies significantly inhibited the entry of SARS-CoV-2 into the cells. Further studies using mouse NIH3T3 cells expressing human ACE2, integrin αv, integrin β1, and/or integrin αvβ1 suggest that integrin αvβ1 was unable to function as an independent receptor but could significantly facilitate the cellular entry of SASR-CoV-2. Finally, we observed that the Omicron exhibited a significant increase in the ACE2-mediated viral entry. Our findings may enhance our understanding of the pathogenesis of SARS-CoV-2 infection and offer potential therapeutic target for COVID-19. While h2+ or Mg2+ dependent cell adhesion factor that medicate the recognition of extracellular matrix and cell-surface ligands (Integrin (ITG) is a heterodimer transmembrane protein consisting of α subunit (containing 18 subtypes) and β subunit (containing 8 subtypes), and a Ca ligands . The I d ligands . In addi ligands . Computa ligands . Moreove ligands , For exa ligands , whereas ligands . ACE2 ex ligands . Integri ligands . While s ligands , others  ligands . HoweverHere, we examined the expression levels of various RGD-binding integrins in a panel of human cell lines and tested the susceptibility of these cell lines to SARS-CoV-2 using the pseudotyped viral system. We found that integrin αvβ1 was highly enriched in the susceptible cell lines. We performed additional studies and demonstrated that integrin αvβ1 significantly facilitated the ACE2-mediated SARS-CoV-2 entry in human Calu-3 and 293T cells as well as mouse NIH3T3 cells. This study may enhance our understanding of the pathogenesis of COVID-19 and offer potential therapeutic target for COVID-19.22.1https://www.ncbi.nlm.nih.gov/labs/virus/). The lentiviral packaging plasmid psPAX2 was obtained from Huayueyang, China. The pLenti-GFP-luci lentiviral reporter plasmid that expresses GFP and luciferase was purchased from TSINGKE, China. The plasmids of the pLVX-puro, pLVX-puro-ACE2, pLVX-puro-ITGAV, and pLVX-puro-ITGB1 were also obtained from TSINGKE. All plasmid constructs used in this study were confirmed by DNA sequencing.Codon-optimized cDNA encoding the spike protein of SARS-CoV-2 (NCBI: NC_045512), SARS-CoV-2 RGD (403–405)→AAA variant, Gamma (NCBI: MZ477758), Delta (NCBI: MZ377115), Omicron (NCBI: OL672836) and VSV-G was synthesized and cloned into the eukaryotic cell expression vector pcDNA3.1(+) or pCMV3 , respectively. The complete coding sequence (CDS) of the spike protein of SARS-CoV-2 variants were retrieved from the database of NCBI Virus (2.22 in the indicated media and passaged every 3–4 days.The 293T (human embryonic kidney cells), SW620 (human colon cancer cells), IOSE-80 (human ovarian cancer cells), MDA-MB-231 (human breast cancer cells), HepG2 (human liver cancer cells), PANC-1 (human pancreatic cancer cells), HGC-27 , BEAS-2B , Calu-3 (human lung adenocarcinoma cells) and NIH3T3 (mouse embryonic fibroblast cells) were obtained from BeNa culture collection . The cells were maintained in their respective medium  with 10% fetal bovine serum  and 1% Penicillin-Streptomycin (PS) . Cell cultures were maintained in a humidified incubator at 37 °C in 5% COThe stable cell lines of 239T cells with empty vector (239T/vector), 239T cells stably expressing recombinant human ACE2 (293T/hACE2), NIH3T3 cells with empty vector (NIH3T3/vector), and NIH3T3 cells stably expressing recombinant human ACE2 (NIH3T3/hACE2) were established using the lentivirus system, respectively . All the2.3The pseudotyped virions were produced by co-transfection of 293T cells with psPAX2, pLenti-GFP-luci, and plasmids encoding coronavirus spike or VSV-G using Lipo8000  . The sup2.44 cells per well were seeded in a 96-well plate for one day, cells were inoculated with 10μl media containing the pseudotyped virions  overnight. Then the medium was removed with addition of fresh medium, and cells were incubated for another 48 h and analyzed for luciferase activity using the ONE-Glo™ Luciferase Assay System  by a GloMax luminometer . To further investigate the contributions of the receptor usage for viral entry, the cells were incubated with 1 μg/ml or 10 μg/ml of different antibody (Ab) that targets the respective receptor (Supplementary Table 2) at 37 °C for 1 h before inoculation with the pseudotyped virions.For viral entry assay, 2 × 102.5Reverse transcription quantitative real-time PCR (RT-qPCR) was performed to detect the mRNA expression level of the target gene according to the manufacturer's instructions. In brief, total RNA was first extracted using the TRIzol Reagent , which was then converted to complementary DNA (cDNA) using a FastKing RT Kit (with gDNase) . The qPCR was performed using 2X ChamQ Universal SYBR qPCR Master Mix Kit  with the QuantStudio DX Real Time System . All primers used in this study were listed in Supplementary Table 1.2.6@-P Membrane, IPVH00010). After blocked by 5% milk at 4 °C for 2 h, the membranes were blotted with primary antibodies overnight at 4 °C. Following 0.1% Tween-20 (TBST) washing, the membranes were incubated with their respective horseradish peroxidase (HRP) conjugated secondary antibodies for 1 h at room temperature. After washing, the signal was detected with SuperSignalTM West Femto Maximum Sensitivity Substrate /chemiluminescence (ECL) kit  and visualized with Chemiluminescent Reagent (Bio-Rad).Western blotting (WB) was used to detect the protein expression level. Briefly, cell protein lysates were prepared in RIPA buffer  with 1 × protease inhibitor cocktail . After measuring the protein concentration using a BCA kit , 5 × loading buffer  was added. Then these samples were boiled for 10 min and separated in a 10% SDS-PAGE gel and transferred to PVDF/nitrocellulose filter membranes (ImmobilonFor immunoprecipitation (IP) and co-IP assays, 293T cells were seeded into 60 mm culture dish plate and transfected with plasmids of RBD-flag, RBD-RGD (403–405)→AAA-flag, ITGAV, and ITGB1 respectively when cells reached 70–80% confluence. The cell lysate was prepared at 48 h post transfection and the supernatants were collected by centrifugation at 4 °C and quantified by the BCA assay. Briefly, mouse anti-flag antibody and 40 μL protein A/G agarose beads  were added into 600 μL (1 μg/μL) supernatants and then incubated at 4 °C overnight. The beads were washed completely, and the co-IP product was obtained with 60 μL of 2 × SDS-PAGE loading buffer. After incubation for five minutes at 100 °C, 15 μL of the co-IP products was used for western blotting analysis using anti-ITGAV and anti-ITGB1 antibodies.The Flow Cytometry was performed to detect the expression level of cell surface proteins. Briefly, cells were incubated with primary antibodies at 37 °C for 1 h, followed by secondary antibodies for 1 h. Then cells were washed twice with PBS containing 2% FBS and 2 mM EDTA , and analyzed by using a BD Accuri C6 flow cytometer and software . All antibodies were listed in Supplementary Table 2.2.7http://gepia2.cancer-pku.cn/#index) database was used to visualize the gene expression. The SARS-CoV-2 spike glycoprotein structure QHD43416 from I-TASSER (https://www.zhanggroup.org/COVID-19/) based on experimental structure  was used for the modeling of SARS-CoV-2 RGD (403–405)→AAA variant spike (3VI4) and integrin AV (PDB ID:6UJC) were used as the template for building homology model of integrin AVB1. Modeling was performed via the website Swiss Model (https://www.swissmodel) with the default parameters (https://bianca.science.uu.nl/haddock2.4/) with the default parameters. The most reliable docking model was selected by the Z-score according to HADDOCK (6M1D) was calculated using the prime module of Schrodinger software . Differences were considered to be significant when P<0.05 (indicated with one asterisk (*)), P<0.01 (indicated with two asterisks (**)), P<0.001 (indicated with three asterisks (***)).All experiments were done in triplicates, and repeated at least twice or more. The data were analyzed using Prism 5.0 software . Data are expressed as the mean ± standard error of the mean (SEM). Statistical significance between two groups was determined by unpaired two-tailed Student's 33.1P = 9.75E-04), Calu-3 (P = 3.01E-04), and 293T cells (P = 2.22E-05) were permissive to SARS-CoV-2 pseudotyped virus infection in vitro , there was no significant difference in susceptibility between these two cell lines (P = 9.16E-02). However, integrin β1 expression level in Calu-3 was higher than 293T (P = 1.17E-04), indicating the potential promotion effect of integrin β1 on the ACE-2 mediated cellular entry of SARS-CoV-2. Taken together, our data suggest that integrin αvβ1 may promote the SARS-CoV-2 infection.It has been proposed that an integrin recognition motif RGD (403–405) in the SARS-CoV-2 spike protein may mediate the interaction between SARS-CoV-2 RGD and the RGD-dependent binding integrins, such as αvβ1, αvβ3, αvβ5, α5β1, αvβ6, αvβ8, and αIIbβ3 B Yan et. Thus, w3.2P = 2.87E-02; 293T, P = 3.61E-02; 293T/hACE2, P = 3.42E-02). In addition, we performed Co-IP experiments using FLAG-tagged SARS-CoV-2 RBD, FLAG-tagged SARS-CoV-2 RGD (403–405)→AAA variant RBD, ITGAV, and ITGB1. To detect the interaction between the RGD motif in SARS-CoV-2 and integrin αvβ1, we generated the pseudotyped viruses bearing the S protein with the wild type (WT) RGD and the RGD (403–405)→AAA variant, respectively, and examined the infectivity of these two pseudotyped viruses in Calu-3, 293T and 293T/hACE2 cells A. The viTo understand the possible influence of RGD (403–405)→AAA variant on the incorporation of S protein into the pseudotyped virions, we examined the levels of the S and p24 proteins in the virions and the S proteins in the cell lysate by western blot. The results revealed that the full-length S proteins (∼180 kDa) were detected in both the WT RGD and RGD (403–405)→AAA pseudotyped virons, but the level of the mutant RGD was markedly reduced compared with the WT one C. Based 3.3P = 1.78E-02), anti-αv (P = 1.99E-03) and anti-β1 (P = 2.74E-03) antibodies all exhibited significant blocking activities. Surprisingly, the anti-integrin antibodies had more potent inhibitory activities than the anti-ACE2 antibody. Approximately 85% reduction of the luciferase activities in the infected cell with anti-integrin antibodies (10μg/ml) whereas about 50% decrease in the cells treated with anti-ACE2 antibody (10μg/ml). However, the combination of anti-αv and anti-β1 integrin antibodies had no significant effect on the viral entry (P = 6.92E-02). In 293T cells (P = 3.93E-03), anti-αv integrin (P = 1.69E-02), and the combination of anti-αv and anti-β1 integrin antibodies (P = 2.73E-03) significantly inhibited the viral entry. However, the anti-β1 integrin antibody (P = 1.11E-01) did not show significant effect. In 293T/hACE2 cells (P = 2.16E-03), anti-αv (P = 5.73E-04), anti-β1 (P = 3.84E-02), and the combination of anti-αv and anti-β1 integrin antibodies (P = 7.77E-05) all significantly inhibited the viral entry. Of note, all the antibodies showed inhibitory effect in the respective cell lines acted in a dose dependent manner. Moreover, all the antibodies mentioned above had no significant inhibitory effect on the viral entry of the VSVG-pseudotyped viruses. Our results suggest that the anti-αvβ1 integrins may play an important role in the viral entry of SARS-CoV-2 into the susceptible cell lines.To examine the role of αvβ1 integrin in the SARS-CoV-2 entry into host cells, we infected three susceptible cell lines  with the pseudotyped viruses in the presence and absence of anti-αvβ1 integrin antibodies, anti-ACE2 antibody (a positive control), and the isotype control antibody (a negative control), respectively. In Calu-3 cells A, anti-A3T cells B, the anE2 cells C, the an3.4P = 3.94E-03), integrin β1 (P = 7.88E-03) and integrin αvβ1 (P = 7.35E-03) significantly increased the viral entry, whereas there was no significant change in the 293T cells expressing integrin αv (P = 4.06E-03), the NIH3T3 cells expressing integrin αv, integrin β1 or integrin αvβ1 had no significant effect on the viral entry, suggesting that integrin αv, integrin β1 or integrin αvβ1 could not function as an independent receptor for the viral entry of SARS-CoV-2 (P = 3.58E-02) and NIH3T3/hACE2-ITGAVB1 (P = 1.61E-02) cells compared with the WT one, respectively (P = 8.60E-03) in entry into 293T/hACE2-ITGAVB1 compared with 293T/hACE2, which was consistent with the findings in NIH3T3/ hACE2-ITGAVB1 cells . In addition, α5β1 and αvβ5 integrins have been shown to be involved in SARS-CoV-2 infection in the respiratory system , suggest3), and the mutation of an amino acid residue to Ala-has been widely used to investigate the function of the mutant protein in many studies (The RGD motif in the spike protein of SARS-CoV-2 has been suggested to be essential element for virus internalization . Here, t studies . Interes studies , suggestThere are some discrepancies between 293T and NIH3T3 cells regarding the contributions of integrin αvβ1 to the ACE2-meddiated viral entry. For instance, there was no significant difference in the viral entry efficiency between 293T/hACE2 and 293T/hACE2+ integrin αvβ1 B, whereaOur data suggested that integrin αvβ1 could not function as an independent receptor for the cellular entry of SARS-CoV-2, but it can serve as a facilitator for the ACE2-mediated viral entry through the interaction with the RGD motif in the spike protein of SARS-CoV-2. Our finding that the Omicron exhibited a significant increase in the ACE2-mediated viral entry is consistent with results from the inhibition study of the integrin signaling by Huntington KE et al. . In addiTo our best knowledge, this is the first study reporting that integrin αvβ1 may significantly facilitate ACE2-mediated entry of SARS-CoV-2. Our findings may not only help to dissect the cellular factors involved in the viral entry of SARS-CoV-2, but may also enhance our understanding of the pathogenesis of COVID-19.Zeqiong Cai: Methodology, Writing – original draft. Han Bai: Methodology, Writing – original draft. Doudou Ren: Methodology, Writing – original draft. Biyun Xue: Methodology. Yijia Liu: Methodology. Tian Gong: Methodology. Xuan Zhang: Methodology. Peng Zhang: Methodology. Junsheng Zhu: Methodology. Binyin Shi: Supervision, Funding acquisition. Chengsheng Zhang: Conceptualization, Methodology, Resources, Writing – original draft, Writing – review & editing, Supervision, Funding acquisition, Project administration.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Cellvibrio sp. KY-GH-1, were expressed in an Escherichia coli system and their activities were compared. Only GH16B , with N-terminal 22-amino acid signal sequence, was secreted into the culture supernatant and demonstrated a robust endolytic agarose hydrolyzing activity for producing neoagarotetraose (NA4) and neoagarohexaose (NA6) as end products. The optimal temperature and pH for the enzyme activity were 50 °C and 7.0, respectively. The enzyme was stable up to 50 °C and over a pH range of 5.0–8.0. The kinetic parameters, including Km, Vmax, kcat, and kcat/Km, of GH16B β-agarases for agarose were 14.40 mg/mL, 542.0 U/mg, 576.3 s−1, and 4.80 × 106 s−1 M−1, respectively. The addition of 1 mM MnCl2 and 15 mM tris(2-carboxyethyl)phosphine enhanced the enzymatic activity. When agarose or neoagaro-oligosaccharides were used as substrates, the end products of enzymatic catalysis were NA4 and NA6, whereas agaropentaose was produced along with NA4 and NA6 when agaro-oligosaccharides were used as substrates. Treatment of 9%[w/v] melted agarose with the enzyme (1.6 µg/mL) under continuous magnetic stirring at 50 °C for 14 h resulted in efficient agarose liquefaction into NA4 and NA6. Purification of NA4 and NA6 from the enzymatic hydrolysate  via Sephadex G-15 column chromatography yielded ~ 650 mg NA4/~ 900 mg NA6 . These findings suggest that the recombinant thermostable GH16B β-agarase is useful for agarose liquefaction to produce NA4 and NA6.Four GH16 family β-agarases , originated from an agarolytic bacterium  Escherichia coli.Thermostable GH16B β-agarase was expressed extracellularly in Complete enzymatic hydrolysis into NA4 and NA6 was achieved using 9%[w/v] melted agarose.The enzyme is useful for producing NAOSs  via agarose liquefaction.NA4 and NA6 in the hydrolysates were purified via Sephadex G-15 column chromatography. Gelidium, Gracilariais, and Gelidiella, contains two major components agarose and agaropectin. Agarose is a non-ionic linear polysaccharide consisting of the repeating unit of neoagarobiose disaccharide, in which 3,6-anhydro-α-l-galactose is α-1,3-linked to β-d-galactose and resulting neoagarobiose is linked by β-1,4 glycosidic bond between β-d-galactose and 3,6-anhydro-α-l-galactose  , we recently reported the genes encoding agarases  from Cellvibrio sp. KY-GH-1, and their enzymatic activities were compared. The isozyme with the highest endolytic β-agarase activity and its efficiency for NA4/NA6 production via agarose liquefaction were also investigated.In this study, an d-galactose, incomplete Freund’s adjuvant, isopropyl β-d-1-thiogalactopyranoside (IPTG), kanamycin, naphthoresorcinol, sodium dodecyl sulfate (SDS), and tris(2-carboxyethyl)phosphine (TCEP) were obtained from Sigma-Aldrich . Restriction enzymes and T4 ligase were obtained from Roche . The pET-30a vector for expressing C-terminal 6× His-tagged protein and Immobilon-P membrane were purchased from EMD Millipore Corporation . A standard NAOS mixture (NA2–NA18) was provided by Dr. Sang-Hyeon Lee  pLysS was obtained from Novagen .Agarose, Micro BCA protein assay kit, Ni-NTA resin, and PageRuler prestained protein ladder were purchased from ThermoFisher Scientific . Ammonium sulfate, chloramphenicol, complete Freund’s adjuvant, Coomassie brilliant blue (CBB) R-250, 3,5-dinitrosalicylic acid (DNS), ethylenediamine tetraacetic acid (EDTA), Cellvibrio sp. KY-GH-1 strain were amplified using polymerase chain reaction (PCR) with the following primers: NdeI-forward primers ; BamHI-forward primer (5′-CGGGATCCATG-AAAAAGCATATTTCATGCTG-3′ for GH16A); XhoI-reverse primers ; and HindIII-reverse primer (5′-CGAAGCTTGAGTGAGCCAACTCGAGTAA-3′ for GH16A). The PCR products were digested with appropriate restriction enzyme sets before being ligated into the pET-30a vector with T4 ligase. The recombinant pET-30a plasmids were transformed into E. coli BL21 (DE3) pLysS. After overnight incubation at 30 °C on Luria broth (LB) plates supplemented with 50 µg/mL chloramphenicol and 25 µg/mL kanamycin, the transformants were selected.Four GH16 β-agarase genes  in the genomic DNA of 600) of the culture reached 0.5–0.6, 0.5 mM IPTG was added to induce protein expression. Then, the culture was incubated at 20 °C for 20 h.The expression of each recombinant β-agarase in the transformant was induced as previously described  pLysS transformants harboring the recombinant pET-30a plasmid containing GH16A, GH16B, GH16C, or GH16D were spotted onto LB agarose plates  and then incubated for 3 days at 37 °C. The plates were stained with Lugol’s iodine solution at 25 °C.http://www.ncbi.nlm.nih.gov). Multiple alignment of the aa sequences was performed using Clustal X program  and cell pellets were separated. Cell pellets were suspended in 40 mM Tris-HCl buffer (pH 8.0), sonicated using 50 short bursts of 2 s each, extracted at 4 °C for 30 min, and fractionated into three intracellular fractions , as previously described , obtained following centrifugation , and the mixture was stirred overnight at 4 °C to precipitate the His-tagged GH16B β-agarase. The precipitate was collected via centrifugation , dissolved in 10 mL of 10 mM Tris-HCl buffer (pH 7.5), and dialyzed at 4 °C for 3 h. The dialyzed recombinant protein was purified via immobilized metal affinity chromatography with Ni-NTA resin. The purified enzyme was lyophilized to dryness.The supernatant of transformed To prepare mouse polyclonal antibody specific for GH16B β-agarase, purified recombinant GH16B β-agarase solution  was mixed with an equal volume of complete Freund’s adjuvant, and the mixture (20 µg/100 µL) was intraperitoneally injected into mice. A purified GH16B β-agarase solution was mixed with an equal volume of incomplete Freund’s adjuvant and injected intraperitoneally into the mice every 10 day for secondary, tertiary, and quaternary immunization. Five days after quaternary immunization, the mice were bled. C57BL/6J male mice (6-weeks-old) were purchased from the JA Bio  and maintained at the Animal Resources Center in Kyungpook National University  under specific pathogen-free conditions.GH16B β-agarase activity was measured as previously described Miller . One hunKm and Vmax values. Effects of metal ions , reducing agent (TCEP), and a chelator (EDTA) on the enzymatic activity were examined by adding these reagents at indicated concentrations to the standard assay mixture.The effect of temperature on the enzymatic activity was measured using 50 mM Mcllvaine buffer buffer (pH 7.0) for 30 min over a temperature range of 25–70 °C. The effect of pH on the enzymatic activity was measured using 50 mM McIlvaine (pH 4.0–7.0) and 50 mM Tris–HCl (pH 7.0–10.0) buffer. For temperature and pH stability assays, aliquots of enzyme were preincubated over a range of temperatures and pH for indicated time, and the relative enzymatic activity was measured. The kinetic parameters of GH16B β-agarase were determined using a known amount of the enzyme and substrate solution . The Lineweaver–Burk equation, generated via GraphPad Prism 8 statistical package , was used to calculate n-butanol-ethanol-H2O (3:2:2[v/v]). To detect oligosaccharides, a visualization solution  was sprayed on the plates, followed by heating at 80 °C. L-AHG , which were developed with To analyze the hydrolytic products of agarose or AOS produced via enzymatic reaction, MALDI–TOF/TOF MS analysis was performed in the positive ion reflectron mode using Autoflex max , as described elsewhere  with continuous magnetic stirring, followed by freeze–drying. The dried samples were dissolved in deionized (DI) water and fractionated via size-exclusion chromatography using Sephadex G-15 column (I.D. 1.3 × 120 cm). Two milliliters of fractions eluting in DI water were collected. TLC was used to identify fractions containing NA4 and NA6, which were then collected and lyophilized to dryness.To purify NA4 and NA6 from the hydrolytic products of agarose, 9%[w/v] melted agarose (20 mL) was treated with GH16B β-agarase under optimal conditions .Unless otherwise specified, the data represent at least three independent experiments and are expressed as the mean ± standard deviation (n ≤ 3). Statistical analyses were performed using Student’s Cellvibrio sp. KY-GH-1, revealed four GH16 β-agarase genes     also appeared to contain the signal sequence at their N-terminal, supporting the possibility that these GH16 family β-agarases are produced in agarolytic Cellvibrio species as extracellular enzymes to degrade agarose. However, the overall aa sequence of Cellvibrio sp. KY-GH-1 GH16B β-agarase (GenBank accession no. WP_151030830.1) showed 100% identity with that of Cellvibrio sp. KY-YJ-3 GH16B β-agarase (GenBank accession no. WP_151030830.1). The GH16B β-agarase sequence of KY-GH-1 also shared 98.8%, 96.0%, 95.0%, and 79.6% similarity with that of nonmarine agarolytic bacteria, such as Cellvibrio sp. pealriver, Cellvibrio sp. OA-2007, Cellvibrio sp. BR, and uncultured bacterium (GenBank accession no. AAP49316.1), respectively, indicating a high degree of homology among Cellvibrio GH16B β-agarases. The GH16B β-agarase sequence of KY-GH-1 shared 53.1%, 52.1%, 50.9%, and 50.8% similarity with that of marine agarolytic bacteria, including Pseudomonas sp. ND137 (GenBank accession no. BAD88713.1) (Aoki and Kamei Marinimicrobium agarilyticum (GenBank accession no. WP_036188483.1), Gilvimarinus chinensis (GenBank accession no. WP_020208740.1), and Simiduia agarivorans (GenBank accession no. WP_015048661.1), respectively.The aa sequence of GH16B β-agarase from Cellvibrio sp. KY-GH-1/KY-YJ-3 is more closely associated with that of Cellvibrio sp. pealriver, Cellvibrio sp. OA-2007, Cellvibrio sp. BR, and uncultured bacterium isolated from nonmarine sources than with that of Pseudomonas sp. ND137, M. agarilyticum, G. chinensis, and S. agarivorans isolated from marine sources   and NA6 ([M+Na]+: 959) corresponded to those calculated from their molecular formulas, as previously described , NA4, and NA6 were produced as end products of the reaction . The complete hydrolysis of agarose into NA4 and NA6 was achieved up to 9.0%[w/v] agarose . The hydrolysis products of agarose  were analyzed using TLC after 14 h of treatment with GH16B β-agarase (1.6 µg/mL) under optimal reaction conditions , which encode GH16 β-agarase isozymes that hydrolyze agarose into NA4/NA6 . However, the mechanism underlying this phenomenon remains unknown.Previously, we observed that the genome of Alignment of the GH16B β-agarase sequence with its top nine homologs in the NCBI GenBank database revealed that GH16B β-agarase homologs exist in all agar-degrading marine and nonmarine bacteria, suggesting that GH16B β-agarase is critical for the bacterial agar-degrading enzymatic machinery. Furthermore, as the enzymatic properties of none of these nine homologs were examined, the current data on GH16B β-agarase may provide insights into GH16 family β-agarases with significant homology to GH16B β-agarase.Km and Vmax values (14.40 mg/mL and 542.0 U/mg) for agarose liquefaction to those of other reported GH16 β-agarases, including GH16 β-agarase (2.59 mg/mL and 275.48 U/mg) from Cellulophaga omnivescoria W5C  for agarose liquefaction.KY-GH-1 GH16B β-agarase showed comparable 2+ and Na+ ions , treatment with 1.6 µg/mL GH16B β-agarase for 14 h under continuous magnetic stirring efficiently hydrolyzed up to 9.0%[w/v] melted agarose into NA4 and NA6. When NA4 and NA6 were purified from the hydrolysates of 9%[w/v] agarose via size-exclusion column chromatography, the recovery of NA4 and NA6 was ~ 85% of the theoretical maximum yield, suggesting that recombinant GH16B β-agarase may be a robust enzyme for industrial use in enzymatic liquefaction of agarose and subsequent mass production of NA4/NA6.Under optimal reaction conditions (1 mM MnClE. coli expression system, could efficiently hydrolyze up to 9%[w/v] agarose in an endo-acting manner to produce NA4 and NA6. The optimal temperature and pH for the enzymatic activity for agarose hydrolysis were 40–50 °C and 7.0, respectively. The enzyme was stable at a temperature of up to 50 °C and pH of 5.0–8.0. In the presence of 1 mM MnCl2 and 15 mM TCEP, the enzymatic activity was enhanced by 2.6-fold. The Km, Vmax, kcat, and kcat/Km values for agarose liquefaction were 14.40 mg/mL, 542.0 U/mg, 576.3 s−1, and 4.80 × 106 s−1 M−1, respectively. The thermal stability and endolytic action of GH16B β-agarase suggest that it is useful for mass production of NA4 and NA6 via one-step agarose liquefaction at 50 °C, which is higher than the gelling temperature of melted agarose. Alternatively, agarose liquefaction using GH16B β-agarase can be combined with a potent exolytic GH50 family β-agarase for efficient agarose degradation into NA2 or can be combined with the exolytic GH50 family β-agarase as well as α-NABH for efficient agarose degradation into the monosaccharides L-AHG and D-Gal. Finally, because the enzymatic properties of none of the nine GH16B β-agarases with 50.8−98.8% homology have been examined to date, current data on GH16B β-agarase from Cellvibrio sp. KY-GH-1 may provide insights into β-agarases exhibiting significant homology with GH16B β-agarase.In summary, the recombinant GH16B β-agarase. which was expressed extracellularly in the


The 8-aminoquinoline moiety is protonated at both the amino and the ring N atoms. In the crystal, the cations and anions are connected by intermolecular N—H⋯Cl and C—H⋯Cl hydrogen bonds, forming sheets parallel to (001). Adjacent sheets are linked through π–π interactions involving the pyridine and arene rings of the 8-azaniumylquinolinium dication. Hirshfeld surface analysis indicates that the most important contributions to the crystal packing are from H⋯Cl (48.1%), H⋯H (19.9%), H⋯C/C⋯H (14.3%) (involving the cations) and H⋯Cl (82.6%) (involving the anions) interactions.The reaction of 8-aminoquinoline, zinc chloride and hydrochloric acid in ethanol yielded the title salt, (C These interactions can be seen as spikes with a sharp tip. H⋯H, H⋯C/C⋯H and C⋯C interactions in the dication, and C⋯Cl and Cl⋯Cl interactions in the dianion follow with contributions of 19.9, 14.3, 6.7, 7.4 and 5.4%, respectively  and added to a solution of ZnCl2  in 10 ml of the same ethanol/HCl mixed solvent. The mixture was heated under reflux and stirred for 30 min. A pale-yellow crystalline product was obtained at room temperature after 6 d by slow solvent evaporation .Commercially available starting materials were used without further purification. 8-Aminoquinoline  was dissolved in 10 ml of an ethanol/HCl mixture (9:1 6.Uiso(H) = 1.2Ueq(C) and C—H = 0.93 Å for aromatic H atoms. Both the amino and the ring N-bound H atoms were located in a difference Fourier map and refined with bond-length restraints of 0.89 (1) and 0.86 (1) Å, respectively.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989023007466/wm5692sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989023007466/wm5692Isup2.hklStructure factors: contains datablock(s) I. DOI: 2290822CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


C(7) chain extending along the a-axis direction.The molecules of the title compound are linked by N—H⋯N hydrogen bonds, generating a  17H14Cl2N2, the molecule exists in an E configuration with respect to the C=N bond of the Schiff base fragment. The dihedral angle between the indole ring system and the benzene ring is 80.86 (12)°. In the crystal, molecules are connected by N—H⋯N hydrogen bonds, generating a C(7) chain extending along the a-axis direction. No aromatic π–π stacking occurs but weak C—H⋯π interactions are observed.In the title compound, C These het al., 2016H-indol-3-yl)-ethyl]-amine skeleton yielded the hits 1-(anthracen-9-yl)-N-[2-(1H- indol-3-yl)ethyl]methanimine ]-5-methylphenol -[1-cyano-1-cyclohexyl-2-(1H-indol-yl)ethyl]iminomethyl} benzonitrile  ethyl]imino}-2-(4-methoxyphenyl)ethan-1-one ethan-1-amine (0.01 mmol) and 2,4-dichlorobenzaldehyde (0.01 mmol), which were taken separately, dissolved in 40 ml of ethanol, then mixed, and heated on a water bath for one h, then kept for crystallization. After a few days, colourless plate-shaped crystals were obtained.The title compound was synthesized by condensing tryptamine, 2- global, I. DOI: 10.1107/S2414314623007800/hb4446Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623007800/hb4446Isup3.cmlSupporting information file. DOI: 2290063CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


They were referred for PET imaging with the suspected diagnosis of a 4‐repeat (4R)‐tauopathy Figure 18F] FDG‐PET revealed a corticobasal degeneration‐like bilateral reduction of cerebral glucose metabolism in frontoparietal cortical areas (arrowheads) and in the thalamus.18F]PM‐PBB318F]PM‐PBB3 binding in patient #2 questioned the diagnosis of a 4R‐tau related corticobasal syndrome . Diagnosis of Creutzfeld‐Jakob disease in this patient was made via 14–3‐3 and PrPSc aggregation assays. In conclusion, [18F]PM‐PBB3‐PET reveals clinical and [18F] FDG‐PET mimics of 4R‐tau CBS caused by Creutzfeld‐Jakob disease.This image depicts 8F FDG an(1) Research project: A. Conception, B. Organization, C. Execution; (2) Statistical analysis: A. Design, B. Execution, C. Review and critique; (3) Manuscript: A. Writing of the first draft, B. Review and critique.N.S.: 1A, B, 1C, 2C, 3A.G.B.: 1C, 2B, 2C, 3B.P.T.M.: 1B, 2A, 2C, 3B.M.R.: 1B, 2C, 3B.J.B.: 1C, 2A, 2C, 3A.Ethical Compliance Statement: The authors confirm that the approval of an institutional review board was not required for this work. Both patients gave written informed consent for the scientific use of imaging and clinical data.Funding Sources and Conflicts of Interest: Nils Schröter, Berta‐Ottenstein‐Programme for Clinician Scientists, Faculty of Medicine, University of Freiburg. The authors have no potential conflict of interest to report. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines.Financial Disclosures for the Previous 12 Months: The authors have nothing to disclose.


The p53 isoform Δ133p53β is known to be associated with cancers driven by inflammation. Many of the features associated with the development of inflammation in rheumatoid arthritis (RA) parallel those evident in cancer progression. However, the role of this isoform in RA has not yet been explored. The aim of this study was to determine whether Δ133p53β is driving aggressive disease in RA.Using RA patient synovia, we carried out RT-qPCR and RNAScope-ISH to determine both protein and mRNA levels of Δ133p53 and p53. We also used IHC to determine the location and type of cells with elevated levels of Δ133p53β. Plasma cytokines were also measured using a BioPlex cytokine panel and data analysed by the Milliplex Analyst software.Δ133TP53 and TP53β mRNA. Located in perivascular regions of synovial sub-lining and surrounding ectopic lymphoid structures (ELS) were a subset of cells with high levels of CD90, a marker of ‘activated fibroblasts’ together with elevated levels of Δ133p53β.Elevated levels of pro-inflammatory plasma cytokines were associated with synovia from RA patients displaying extensive tissue inflammation, increased immune cell infiltration and the highest levels of + synovial fibroblasts leads to an increase in cytokine and chemokine expression and the recruitment of proinflammatory cells into the synovial joint, creating a persistently inflamed environment. Our results show that dysregulated expression of Δ133p53β could represent one of the early triggers in the immunopathogenesis of RA and actively perpetuates chronic synovial inflammation. Therefore, Δ133p53β could be used as a biomarker to identify RA patients more likely to develop aggressive disease who might benefit from targeted therapy to cytokines such as IL-6.Induction of Δ133p53β in CD90The online version contains supplementary material available at 10.1186/s13075-023-03040-8. Rheumatoid arthritis (RA) is a systemic autoimmune disease characterised by chronic inflammation of joint synovial tissue, resulting in destructive, debilitating disease with recognised systemic involvement . Its aetTP53. Typically, mutations in the TP53 gene, or aberrations in molecular pathways affecting p53 protein expression and regulation, lead to unchecked proliferation and tumour growth ; [1: <5]; [2: 5-10]; [3: >10]. (ii) Clusters: [0: negative]; [1: low]; [2: medium]; [3: high]. (iii) Scattered: [0: not present]; [1: <10%]; [2: 10-50%]; [3: >50%]. T cell (CD3): ‘Clusters’ and ‘Scattered’ as above for B cells. Macrophages: [0: negative]; [1: <10% low]; [2: 10-50% medium]; [3: >50% high] [28]. Synovia were classified into 3 pre-defined histopathological subtypes: (1) Follicular: predominantly lymphoid with ELS; (2) Diffuse: dominated by myeloid cells and T lymphocytes; and (3) Pauci-immune: fibroblast dominated, with minimal inflammatory infiltrate [Fig. S3. Western Blot for isoform specific antibodies. A Western blot using the anti-p53β antibody 79.3 (sourced from JC Bourdon lab) on total protein of PC-3 cells transfected with either control plasmid (Empty Vector) or a plasmid expressing either Δ133p53α, Δ133p53β or Δ133p53γ isoforms respectively. B Western blot using KJCA133, an anti-Δ133p53 antibody (sourced from JC Bourdon lab) on total protein of PC-3 cells transfected with either control plasmid (Empty Vector) or a plasmid expressing Δ133p53γ. Fig. S4. Distribution of mRNA expression levels of FL/Δ40TP53_T1, FL/Δ40TP53_T2, Δ133TP53, TP53α, TP53β in OA and RA synovial tissue. Dots represent data from individuals in each group. Lines represent mean and SD. Significance was determined using unpaired t-test with Welch’s correction. *p < 0.05, **p < 0.01, ***p < 0.001. Fig. S5. p53β protein co-localises with CD55+ fibroblast-like synoviocytes (FLS), CD68+ macrophage-like synoviocytes (MLS) and macrophages, and CD90+ cells. Panels 1 through to 4 describe panels left to right. A FLS are CD55+; MLS and macrophages (M) are CD68+B p53β expressing cells , CD55+/ CD68+ expressing cells , Merged (panel 3) showing co-localisation of p53β+ with either CD55+ or CD68+ cells (yellow) and nuclei (blue). 400x magnification; scale bar 50µm. C p53β is highly expressed in CD90+ cells surrounding ELS (top row). p53β is expressed in cells that are CD90+ that resemble plasma cells  and adjacent to p53β+ endothelium  and in endothelial cells . CD90+ and vWF+ cells in endothelial and sub-endothelial layer (bottom row) D The percentage of CD55+, CD68+, and CD90+ cells that also co-express p53β. Each dot represents results from each individual image field and a minimum of 100 cells. The lines represent the mean and SD. Significance was determined using unpaired t test with Welch’s correction . CD90+/p53β+ vs CD68+/p53β+, p<0.0001; CD90+/p53β+ vs CD55+/p53β+, p<0.0001; CD68+/p53β+ vs CD55+ p53β+, p<0.0004. Fig. S6. MCP-1, MIP-1a, MIP-1b and RANTES were elevated in the serum from Δ122p53+/- (blue) compared to p53+/- (red) mice. The bars represent the concentration of the individual cytokines from 4 pooled serum samples. The error bars represent 97% CI. Significance was determined using unpaired t-test with Welch’s correction; *p < 0.05.filtrate , 37. FigAdditional file 2:Table S1. Correlations of Synovial TP53 Transcript Expression, Select Gene Expression and Plasma Cytokine Measures. Shown are Spearman correlation r values between transcript levels of various p53 isoforms and select inflammatory genes present in synovial tissue and with measures of plasma cytokine. Significant correlations are shown in bold with asterisks indicating level of significance: * p<0.05; ** p<0.01; ***p<0.001; and ****p<0.0001.


In the published article, there was an error. The molecular weights of “GF9-HFn/Str, HFn, GF9-HFn and GF9-HFn + MMP2” in the original article were wrong. The molecular weights of “GF9-HFn/Str, HFn and GF9-HFn” in the original article were displayed as “36 kD, 33.1 kD and 36 kD”. But the correct size of “GF9-HFn/Str is 40 kD”, the correct size of “HFn is 35 kD” and the correct size of “GF9-HFn is 40 kD”.Abstract section.In the published article, there was an error in “The size of GF9-HFn/Str is 36 kD”.The corrected sentence appears below:“The size of GF9-HFn/Str is 40 kD”.Results section titled The evaluation of ferritin-based codelivery nanoparticle.In the published article, there was an error in Figure 1A)”.“We found that the size of GF9-HFn/Str (36 kD) is bigger than HFn ”.“We found that the size of GF9-HFn/Str (40 kD) is bigger than HFn ”.“Then, SDS-PAGE assessed the protein molecules. Compared with GF9-HFn (36 kD), the cells added MMP2 enzyme separated the band of GF9-HFn + MMP2 (36 kD) into two bands, which are HFn (33.1 kD) and GF9 (2.9 kD) ”.“Then, SDS-PAGE assessed the protein molecules. Compared with GF9-HFn (40 kD), the cells added MMP2 enzyme separated the band of GF9-HFn + MMP2 into two bands, which are HFn (35 kD) and GF9 (2.9 kD) (The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.