Z)-2-(benzylamino)-5-benzylidenethiazol-4(5H)-one (BABT) derivatives were designed as tyrosinase inhibitors based on the structure of MHY2081, using a simplified approach. Of the 14 BABT derivatives synthesized, two derivatives ((Z)-2-(benzylamino)-5-(3-hydroxy-4-methoxybenzylidene)thiazol-4(5H)-one [7] and (Z)-2-(benzylamino)-5-thiazol-4(5H)-one [8]) showed more potent mushroom tyrosinase inhibitory activities than kojic acid, regardless of the substrate used; in particular, compound 8 was 106-fold more potent than kojic acid when l-tyrosine was used as the substrate. Analysis of Lineweaver–Burk plots for 7 and 8 indicated that they were competitive inhibitors, which was confirmed via in silico docking. In experiments using B16F10 cells, 8 exerted a greater ability to inhibit melanin production than kojic acid, and it inhibited cellular tyrosinase activity in a concentration-dependent manner, indicating that the anti-melanogenic effect of 8 is attributable to its ability to inhibit tyrosinase. In addition, 8 exhibited strong antioxidant activity to scavenge 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals and peroxynitrite and inhibited the expression of melanogenesis-associated proteins . These results suggest that BABT derivative 8 is a promising candidate for the treatment of hyperpigmentation-related diseases, owing to its inhibition of melanogenesis-associated protein expression, direct tyrosinase inhibition, and antioxidant activity.In this study, ( As observed in previous studies [Z)-2-(benzylamino)-5-thiazol-4(5H)-one [10], (Z)-2-(benzylamino)-5-thiazol-4(5H)-one [11], and (Z)-2-(benzylamino)-5-thiazol-4(5H)-one [14]) exhibited little-to-no mushroom tyrosinase inhibitory activity. The introduction of two methoxyl ((Z)-2-(benzylamino)-5-thiazol-4(5H)-one [12]) or two t-butyl ((Z)-2-(benzylamino)-5-thiazol-4(5H)-one [13]) groups at positions 3 and 5 on the β-phenyl ring of derivative 1 also decreased its tyrosinase inhibitory activity . Similarly, the insertion of an alkoxyl group (methoxyl or ethoxyl) at position 3 on the β-phenyl ring of derivative 1 led to low tyrosinase inhibition . Exchanging the two substituents (3-OMe and 4-OH) on the β-phenyl ring in derivative (Z)-2-(benzylamino)-5-(4-hydroxy-3-methoxybenzylidene)thiazol-4(5H)-one (5) resulted in 14.8-fold stronger tyrosinase inhibitory activity ((Z)-2-(benzylamino)-5-(3-hydroxy-4-methoxybenzylidene)thiazol-4(5H)-one [7]: IC50 = 10.0 ± 0.90 µM). Notably, an additional hydroxyl group at position 3 on the β-phenyl ring of derivative 1 reduced its tyrosinase inhibitory activity, but an additional hydroxyl group at position 2 on the β-phenyl ring of derivative 1 drastically increased its tyrosinase inhibitory activity ((Z)-2-(benzylamino)-5-thiazol-4(5H)-one [8]: IC50 = 0.27 ± 0.03 µM; (Z)-2-(benzylamino)-5-thiazol-4(5H)-one [9]: IC50 > 300 µM). When the two phenolic hydroxyl substituents of each derivative (8 and 9) were transformed into fluorine substituents, their tyrosinase inhibitory activities were drastically reduced ((Z)-2-(benzylamino)-5-thiazol-4(5H)-one [3]: IC50 > 300 µM) or very weak ((Z)-2-(benzylamino)-5-thiazol-4(5H)-one [4]: IC50 = 288.7 ± 14.72 µM). A hydroxyl substituent can act as a hydrogen bond donor and acceptor, whereas a fluorine substituent can only act as a hydrogen bond acceptor. Chemical structural differences between derivatives 3 and 8 and their tyrosinase inhibitory activities indicate that substituents at positions 2 and 4 of the β-phenyl ring of the PUSC template play important roles in its tyrosinase inhibitory activity. In particular, their ability to act as hydrogen bond donors has a decisive and significant effect on tyrosinase inhibition.When  studies ,23, the l-dopa was used as a substrate for mushroom tyrosinase, the tested compounds generally showed higher IC50 values than those of tested compounds measured in the presence of l-tyrosine. BABT derivative 1 with a 4-hydroxyl substituent had a 2-fold higher IC50 value (65.1 ± 8.29 µM) compared to the case where l-tyrosine was used. The IC50 value of kojic acid (IC50 = 20.1 ± 0.46 µM) in the presence of l-dopa was similar to that observed in the presence of l-tyrosine. The conversion of 4-OH on the β-phenyl ring of 1 into a methoxy substituent decreased the tyrosinase inhibitory activity by 3.7-fold (2: IC50 = 239.4 ± 2.38 µM), indicating that the role of the 4-substituent as a hydrogen donor is important in conferring tyrosinase inhibitory activity. As observed in the results where l-tyrosine was used as a substrate for mushroom tyrosinase, derivatives  without any hydroxyl substituent on the β-phenyl ring of the PUSC template showed no or weak tyrosinase inhibitory activities. Additional alkoxyl (OMe or OEt) or bulky alkyl (t-butyl) groups at position 3 or positions 3 and 5 on the β-phenyl ring of derivative 1 greatly reduced its tyrosinase inhibitory activity -2-(benzylamino)-5-(3-ethoxy-4-hydroxybenzylidene)thiazol-4(5H)-one [6]: IC50 = 248.1 ± 11.54 µM), and the exchange of 3-OMe and 4-OH substituents of derivative 5 greatly enhanced its tyrosinase inhibitory activity (9: IC50 = 10.3 ± 0.24 µM), which was 2-fold more potent than that of kojic acid. The strongest tyrosinase inhibition was found in derivative 8 with a 2,4-dihydroxyphenyl moiety, and its IC50 value was 1.04 ± 0.05 µM. However, this value was 4-fold higher than that obtained using l-tyrosine as the substrate. Contrary to 8 with two hydroxyl groups at positions 2 and 4, derivative 9 with two hydroxyl groups at positions 3 and 4 had an IC50 value >300 µM, indicating that the positions of the hydroxyl substituents on the β-phenyl ring of the PUSC template sensitively affect the tyrosinase inhibitory activity regardless of the type of substrate (l-dopa or l-tyrosine) used.When 7 with a 3-hydroxy-4-methoxyl substituent and 8 with a 2,4-dihydroxyl substituent showed higher mushroom tyrosinase inhibitory activities than kojic acid, their inhibitory modes of action were investigated using mushroom tyrosinase in the presence of l-dopa. Kinetic studies using Lineweaver–Burk plots were performed using four different concentrations  of the test derivatives (7 and 8) and five different concentrations  of l-dopa. Lineweaver–Burk double reciprocal plots for derivatives 7 and 8 are shown in 7 formed four straight lines that converged on the y-axis. The kinetic results of derivative 8 showed a similar pattern to that of derivative 7. Lineweaver–Burk plots for 8 also formed four straight lines that converged on the y-axis. These kinetic results suggest that the BABT derivatives 7 and 8 compete with their natural substrates, namely, l-tyrosine and l-dopa, at the active site of mushroom tyrosinase.As 7 and 8 were transformed into the corresponding Dixon plots to determine their inhibition constant (Ki) values for the mushroom tyrosinase–inhibitor complex. Dixon plots for 7 and 8 are shown in i values for the mushroom tyrosinase–inhibitor complex were obtained from the x-axis value of each convergence point. The Ki values were 3.1 × 10−6 M for 7 and 8.1 × 10−7 M for 8, indicating that derivative 8 is a stronger mushroom tyrosinase inhibitor than derivative 7.Lineweaver–Burk plots for 7 and 8 showed potent inhibitory effects on mushroom tyrosinase, the binding affinities of these derivatives to the active site of mushroom tyrosinase were predicted using the Schrödinger Suite (release 2021-1) and the mushroom tyrosinase X-ray crystal structure (Protein Data Bank [PDB] ID: 2Y9X). Kojic acid, a well-known tyrosinase inhibitor, was used as a positive control. Tropolone , which binds to the mushroom tyrosinase active site, was deleted, and to the same binding pocket, kojic acid and BABT derivatives 7 and 8 were docked. The binding energies of docked compounds and chemical interactions between compounds 7 and 8 and kojic acid and the amino acid residues present in the active site of mushroom tyrosinase are displayed in 2D and 3D representations in Because derivatives 7 formed two hydrogen bonds between Ser282 and the 4-methoxyl substituent (as a hydrogen bond acceptor) on the β-phenyl ring of the PUSC template, between Gly281 and the 3-hydroxyl substituent (as a hydrogen bond donor) on the β-phenyl ring, and a π–π stacking interaction between His263 and the phenyl ring of the 2-benzylamino group. These interactions provided 7 with a docking score of −4.179 kcal/mol, which is approximately the same as that of kojic acid. Unlike the binding configuration of derivative 7, the β-phenyl ring of the PUSC template of derivative 8 was bound close to copper ions. Derivative 8 created two salt bridge interactions with Cu400 and Cu401, using the 4-hydroxyl substituent of the β-phenyl ring, and made three π–π stacking interactions with His259, His263, and Phe264 using the β-phenyl ring of the PUSC template and the phenyl ring of the benzylamino group. These interactions with the amino acid residues of the mushroom tyrosinase active site provided 8 with a docking score of −6.500 kcal/mol, indicating that 8 binds to the active site of mushroom tyrosinase much more strongly than kojic acid. The different arrangements of derivatives 7 and 8 at the mushroom tyrosinase active site were speculated to have a significant effect on binding affinity. These docking results support the kinetic results that derivatives 7 and 8 competitively inhibit the activity of mushroom tyrosinase.Three interactions were observed in kojic acid A: a meta7 and 8 and kojic acid were determined during 100 ns using Gnuplot software.A molecular dynamics (MD) simulation is typically used to complement and validate the docking results by analyzing the stability and conformational changes of protein–ligand complexes over time . Using G7, 8, and kojic acid) was calculated. The RMSD between tyrosinase C-α and ligands was measured. As a result of the measurement, the RMSD between tyrosinase and ligands had a stable value of 2 nm or less, except for some sections. In the case of 7, it had an overall very low RMSD value of about 0.15 to 0.4 nm. On the other hand, 8 had a higher RMSD value than 7 (0.4 to 0.7 nm), but a lower RMSD value than that of kojic acid. Kojic acid had a similar RMSD value to 7 until 40 ns, but after 40 ns it had an RMSD value approaching 2 nm. RMSD analysis concluded that BABT derivatives 7 and 8 stably bind to tyrosinase, and they show the possibility of showing stronger tyrosinase inhibitory activity than kojic acid.As shown in 7, 8, and kojic acid were similar. This implies that 7 and 8 can have activity similar to that of kojic acid when bound to tyrosinase. In addition, the RMSF values of kojic acid were higher than those of 7 and 8 at tyrosinase residues near positions 125, 150, 200, and 250, indicating that 7 and 8 bind to tyrosinase more strongly than kojic acid. In conclusion, BABT derivatives 7 and 8 are more likely to bind to tyrosinase more tightly than kojic acid and show activity similar to that of kojic acid.The RMSF between tyrosinase C-α and ligands is shown in 7 had more than three hydrogen bonds with tyrosinase at all times, and 8 had fewer hydrogen bonds than 7, but more than three hydrogen bonds after 90 ns  to predict the chemical interactions between each ligand and the amino acid residues of the tyrosinase active site. Unlike derivative 8 and kojic acid, the software did not generate any docking results for derivative 7. The in silico docking simulation results are shown in Due to the lack of a human tyrosinase X-ray crystal structure, we used a previously constructed human tyrosinase homology model ,23. Deri8 was bound close to the metal ions Zn6 and Zn7. Derivative 8 also showed three types of interactions: two salt bridges, a π–π stacking interaction, and two hydrogen bonds. The 4-hydroxyl substituent on the β-phenyl ring generated two salt bridges with Zn6 and Zn7, similar to the 5-hydroxyl substituent on the 4-pyranone ring of kojic acid, and the β-phenyl ring of the PUSC template showed a π–π stacking interaction with His367. The 2-hydroxyl substituent on the β-phenyl ring and amino substituent of the benzylamino group formed hydrogen bonds with Met374 and Glu203, respectively, and these substituents acted as hydrogen bond donors. These interactions with the amino acid residues and Zn metals in the human tyrosinase active site provided 8 with a docking score of −6.841 kcal/mol, which was much lower than that of kojic acid.In kojic acid, three types of interactions were observed: a hydrogen bond with Asn364 via the 2-hydroxymethyl substituent, two π–π stacking interactions with His363 and His367 via the 4-pyranone ring, and two salt bridge interactions with Zn6 and Zn7 via the 5-hydroxyl substituent. These interactions provided kojic acid with a docking score of −4.645 kcal/mol. As in the docking results with mushroom tyrosinase, the β-phenyl ring of the PUSC template of derivative 7 and 8 were found to inhibit cellular tyrosinase and melanin production in B16F10 murine melanoma cells. Before these experiments were performed, the influence of these derivatives on cytotoxicity in B16F10 cells was examined to determine their optimal concentration range for B16F10 cell experiments. The cytotoxicity of derivatives 7 and 8 at six concentrations  was assessed using the EZ-Cytox assay.In addition to exerting potent mushroom tyrosinase inhibitory effects, BABT derivatives 7 was not cytotoxic at all concentrations up to 48 h but showed cytotoxicity at a concentration of 20 µM at 72 h. On the other hand, derivative 8 showed no cytotoxicity until 72 h at all tested concentrations. Thus, only derivative 8 was used for B16F10 cell experiments to determine its effects on cellular tyrosinase activity and melanin production.The viability results measured at three time points  are shown in 8 had potent mushroom tyrosinase inhibitory activity and no significant cytotoxicity in B16F10 cells at concentrations ≤20 µM, its effect on melanin biosynthesis in B16F10 cells was investigated.Since BABT derivative 8 on extracellular and intracellular melanin levels in B16F10 cells, the cells were treated with various concentrations  of derivative 8 for 1 h, in addition to co-treatment with α-MSH (0.5 µM) and 3-isobutyl-1-methylxanthine  to enhance melanin biosynthesis in B16F10 cells. After 72 h of incubation, the effects of 8 on extracellular and intracellular melanin levels were determined by measuring the melanin absorbance in the culture media and cell lysates, respectively, at 405 nm using a microplate reader. Kojic acid (20 µM) was used as the positive control.To investigate the effects of derivative 8 significantly and dose-dependently reduced the extracellular melanin levels enhanced by stimulators to 1.22-, 1.03-, and 1.00-fold at 5, 10, and 20 µM, respectively, compared to those in the untreated control. These results implied that 8 at concentrations ≥10 µM had a strong anti-melanogenic effect, returning the melanin levels increased by α-MSH plus IBMX to the normal levels as observed in the untreated control.8 significantly and dose-dependently reduced the intracellular melanin levels enhanced by stimulators. At 5 µM, 8 showed more potent inhibitory activity on melanin production than kojic acid at 20 µM, and the intracellular melanin levels in cells treated with 8 at 20 µM were lower than those in the untreated control (100%).Intracellular melanin levels were determined by measuring the absorbance of the cell lysates at 405 nm B. Treatm8 was found to exert inhibitory effects on extracellular and intracellular melanin production in B16F10 cells, and tyrosinase acts as a rate-determining enzyme for melanin biosynthesis, the inhibitory effect of 8 on tyrosinase activity was examined using B16F10 cells.Since derivative 8 for 1 h, followed by co-treatment with α-MSH (0.5 µM) and IBMX (200 µM) to induce an increase in cellular tyrosinase activity. The cellular tyrosinase inhibitory activity of 8 was assessed by measuring the absorbance at 475 nm (λmax of dopachrome) using a microplate reader. Kojic acid (20 µM) was used as a positive reference control.To measure cellular tyrosinase activity in B16F10 cells, cells were treated with various concentrations  of derivative 8. Stimulators (α-MSH plus IBMX) enhanced the cellular tyrosinase activity by 3.2-fold compared to that in the untreated control (100%), and treatment with 20 µM kojic acid reduced the cellular tyrosinase activity by 2.9-fold compared to that in the untreated control. Derivative 8 significantly reduced the cellular tyrosinase activity in a dose-dependent manner. The inhibition of cellular tyrosinase activity by 8 at 5 µM was more than that by kojic acid at 20 µM, and 8 at 20 µM decreased the cellular tyrosinase activity by 1.7-fold compared to that in the untreated control. The inhibitory effect of 8 on cellular tyrosinase activity resembled its inhibitory effect on intracellular melanin production, suggesting that the anti-melanogenic effect of 8 may be mainly due to its inhibitory effect on cellular tyrosinase activity.l-tyrosine and l-dopa) [1–14 were evaluated using DPPH and ABTS assays.In addition to the direct inhibition of tyrosinase activity, the antioxidant capacity of BABT derivatives may suppress melanogenesis by inhibiting oxidation processes in melanin biosynthesis, starting from tyrosinase substrates ( l-dopa) ,26. To el-ascorbic acid as a positive reference control. DPPH radical scavenging activities were measured after leaving the mixed samples (test samples of 500 µM plus DPPH solution of 0.18 mM) in the dark for 30 min. To determine the radical scavenging activities, the absorbance of the mixed samples was measured at 517 nm. l-Ascorbic acid exhibited potent DPPH radical scavenging activity with 97% inhibition. Of the 14 BABT derivatives, five BABT derivatives  showed high (76–89% inhibition) and two BABT derivatives (7 and 12) showed moderate  DPPH radical scavenging activities. Derivative 9, with a 3,4-dihydroxyphenyl (catechol) group, showed the most potent DPPH radical scavenging activity with 89% inhibition. Derivative 8, with a 2,4-dihydroxyphenyl (resorcinol) group, also showed strong DPPH radical scavenging activity with 76% inhibition. Derivatives  with no phenolic hydroxyl substituents showed no significant DPPH radical scavenging activity.DPPH is a light-sensitive compound containing free radicals. The DPPH assay is one of the most common methods for evaluating the antioxidant activity of a compound. The effects of BABT derivatives on DPPH radical scavenging activities were evaluated using 8 showed the most potent ABTS radical scavenging activity with 96% inhibition, similar to that of Trolox (99% inhibition). Derivative 9, with a catechol substituent, showed potent ABTS radical scavenging activity with 87% inhibition, and four derivatives  showed moderate ABTS radical scavenging activities with 43–58% inhibition. As observed in the DPPH radical scavenging assay, derivatives without a hydroxyl substituent on the β-phenyl ring of the PUSC template  did not exhibit any ABTS radical scavenging activity. DPPH and ABTS radical scavenging assay results suggest that derivative 8 may suppress melanogenesis via direct tyrosinase inhibition and indirectly via its antioxidant activities, such as DPPH and ABTS radical scavenging activities.An antioxidant assay using ABTS is also commonly used to assess the antioxidant activities of compounds. Donation of one electron from ABTS to potassium persulfate generates the ABTS radical cation. An ABTS radical scavenging activity assay was performed using 100 µM of BABT derivatives or Trolox (a positive reference control) by mixing test samples with ABTS radical cations obtained from a reaction of potassium persulfate and ABTS. ABTS radical scavenging activity was assessed by measuring the optical density at 734 nm after leaving the test sample–ABTS radical cation mixture in the dark for 2 min. 1–14 against ROS and peroxynitrite (ONOO−) were investigated because the antioxidant activities of the compounds may modulate melanogenesis [1–14. BABT derivatives and Trolox, the positive control, were used at a concentration of 20 µM. SIN-1 treatment significantly increased the ROS levels compared to those in the untreated control, and Trolox reduced the ROS levels enhanced by SIN-1. Three derivatives, namely, 1, 2, and 9, with 4-hydroxyphenyl, 4-methoxyphenyl, and 3,4-dihydroxyphenyl groups, respectively, also strongly reduced the ROS levels enhanced by SIN-1, although the extent of reduction was less than that by Trolox. In addition, four derivatives  exhibited moderate ROS scavenging activities. However, derivative 8, which exerted a potent anti-melanogenic effect in B16F10 cells and mushroom tyrosinase inhibitory effect, did not show significant ROS scavenging effect.The scavenging activities of the BABT derivatives ogenesis ,26. To m1–14 were assessed by measuring the fluorescence of rhodamine 123. BABT derivatives and penicillamine, the positive control, were used at a concentration of 20 µM. 8, which showed a potent anti-melanogenic effect in B16F10 cells, scavenged peroxynitrite more potently than did penicillamine. Derivative 1 with a 4-hydroxyphenyl group and derivative 9 with a 3,4-dihydroxyphenyl group showed strong ROS and peroxynitrite scavenging activities.The effects of the BABT derivatives on peroxynitrite scavenging activities were also examined using SIN-1 as an RNS generator and dihydrorhodamine (DHR123) as an ROS indicator. DHR123 was converted to fluorescent rhodamine 123 by peroxynitrite generated by SIN-1. Therefore, the peroxynitrite scavenging activities of 8 may be related to the expression of proteins involved in melanogenesis. For the Western blotting experiment, B16F10 cells were treated with α-MSH (1 µM) plus IBMX (200 µM), leading to a 1.7- and 4.9-fold increase in expression levels of melanogenesis-associated proteins tyrosinase and MITF, compared to those in the untreated control (100%)  . TreatmeGeneral Methods2 or Na/benzophenone). All reactions were performed under a nitrogen atmosphere. The progress of the reaction was monitored using thin-layer chromatography (Silica gel 60 F254). Flash column chromatography using MP Silica  was used to purify the reaction mixture. NMR data were recorded using a Varian Unity AS500 unit  for 125 MHz 13C NMR and 500 MHz 1H NMR. Chemical shifts (δ) were indicated in parts per million (ppm), and coupling constants (J) were indicated in hertz (Hz). The splitting patterns were displayed as singlet (s), broad singlet (brs), doublet (d), broad doublet (brd), triplet (t), broad triplet (brt), quartet (q), doublet of doublets (dd), multiplet (m), and broad multiplet (brm).All reagents and solvents were commercially obtained, and anhydrous solvents were prepared using dry methods (distillation over CaH1H and 13C nuclear magnetic resonance (NMR) spectroscopy and mass (ESI positive and negative modes) spectrometry data of compounds 1–16 can be found in Only the NMR data of the major tautomers are presented below. 15 Synthesis of Compound 3 solution was added to the resultant residue to adjust the pH to 7. The precipitate was filtered and washed with water to obtain 15  as a solid.A solution of chloroacetyl chloride  in acetone (20 mL) was added to a solution of benzylamine  in acetone (80 mL) at 0 °C, and the reaction mixture was stirred for 1 h at the same temperature. The volatiles were removed under reduced pressure, and aqueous NaHCO1H NMR  δ 7.36 , 7.33–7.27 , 6.87 , 4.50 , 4.10 ; 13C NMR  δ 165.9, 137.3, 128.8, 127.8, 127.8, 43.8, 42.6; LRMS (ESI+) m/z 183.99 (M+H)+, calcd 184.05, 206.13 (M+Na)+, calcd 206.03, 208 (M+2+Na)+, calcd 208.03.16 [Synthesis of Compound 16 15  and ammonium thiocyanate  in ethyl alcohol (100 mL) was refluxed for 7 d. After cooling to ambient temperature, volatiles were evaporated under reduced pressure. The resultant precipitate was filtered using dichloromethane, and the filtrate was evaporated. The resulting residue was purified via silica gel column chromatography using dichloromethane and methanol (30:1) as the eluent to give 16  as a solid.A solution of 1H NMR  δ 9.65 , 7.39–7.28 , 4.62 , 3.94 ; 13C NMR  δ 187.5, 181.3, 138.0, 129.0, 128.0, 127.9, 48.3, 40.5; LRMS (ESI+) m/z 207.16 (M+H)+, calcd 207.06, 229.05 (M+Na)+, calcd 229.04.Tautomer ratio = 1:0.2; 1–14General Procedure for the Synthesis of BABT Derivatives 16  and appropriate benzaldehyde (1.1 equiv.) in acetic acid (3 mL) was refluxed for 1–7 d in the presence of sodium acetate . After adding cold water, the precipitate was filtered and washed with water, dichloromethane, ethyl acetate, and/or methanol, and the filter cake was purified via silica column chromatography using dichloromethane and methanol to obtain 1–7 and 9–14 as solids with yields of 36–93%. For the synthesis of 8, 10 was used as the starting material. To a solution of 10  in dichloromethane (2 mL), 1 M BBr3 dichloromethane solution  was added and stirred at room temperature for 3 d. The volatiles were removed under reduced pressure, water was added, and the mixture was filtered. The filter cake was purified via silica gel column chromatography using dichloromethane and methanol (10:1) as the eluent to give 8  as a solid.A solution of 1Compound 1H NMR  δ 10.11 , 7.51 , 7.39 , 7.36–7.33 , 7.29 , 6.88 , 4.70 ; 13C NMR  δ 180.4, 174.2, 159.5, 137.7, 132.0, 130.4, 129.1, 128.2, 128.0, 125.3, 124.9, 116.7, 48.1; LRMS (ESI−) m/z 309.19 (M−H)−, calcd 309.07, LRMS (ESI+) m/z 333.24 (M+Na)+, calcd 333.07.Tautomer ratio = 1:0.2; yield: 93%; 2Compound 1H NMR  δ 10.01 , 7.59 , 7.53 , 7.40–7.35 , 7.31 , 7.08 , 4.74 , 3.81 ; 13C NMR  δ 180.3, 174.1, 160.7, 137.7, 131.7, 129.7, 129.1, 128.2, 128.0, 126.9, 126.2, 115.2, 55.9, 48.1; LRMS (ESI−) m/z 323.27 (M−H)−, calcd 323.03.Tautomer ratio = 1:0.2; yield: 55%; 3Compound 1H NMR  δ 10.17 , 7.58 , 7.57–7.54 , 7.43–7.34 , 7.31–7.26 , 4.74 ; 13C NMR  δ 179.6, 173.9, 163.2 , 161.1 , 137.5, 131.7, 130.3 , 129.1, 128.3, 128.1, 119.7 , 119.3 , 113.1 , 105.3 , 48.4; LRMS (ESI−) m/z 329.29 (M−H)−, calcd 329.06, LRMS (ESI+) m/z 353.23 (M+Na)+, calcd 353.05.Tautomer ratio = 1:0.1; yield: 49%; 4Compound 1H NMR  δ 10.14 , 7.64–7.54 , 7.59 , 7.42–7.33 , 7.29 , 4.73 ; 13C NMR  δ 179.7, 173.9, 150.2 , 150.1 , 137.5, 132.4 , 130.5 , 129.1, 128.2, 128.1, 127.8, 127.6, 126.5 , 118.9 , 48.3; LRMS (ESI−) m/z 329.30 (M−H)−, calcd 329.06, LRMS (ESI+) m/z 353.23 (M+Na)+, calcd 353.05.Tautomer ratio = 1:0.2; yield: 62%; 5Compound 1H NMR  δ 9.95 , 9.72 , 7.54 , 7.41–7.25 , 7.14 , 7.02 , 6.90 , 4.71  3.81 ; 13C NMR  δ 180.3, 174.1, 149.0, 148.3, 137.7, 130.6, 129.0, 128.1, 128.0, 125.8, 125.2, 123.6, 116.5, 114.0, 56.0, 48.1; LRMS (ESI−) m/z 339.23 (M−H)−, calcd 339.08, LRMS (ESI+) m/z 363.22 (M+Na)+, calcd 363.08.Tautomer ratio = 1:0.2; yield: 55%; 6Compound 1H NMR  δ 9.93 , 9.65 , 7.52 , 7.38–7.32 , 7.29 , 7.12 , 7.01 , 6.90 , 4.71 , 4.06 , 1.34 ; 13C NMR  δ 179.9, 173.6, 148.8, 147.0, 137.3, 130.1, 128.6, 127.7, 127.5, 125.3, 124.7, 123.4, 116.1, 114.6, 63.9, 47.6, 14.7; LRMS (ESI+) m/z 355.19 (M+H)+, calcd 355.11, m/z 376.92 (M+Na)+, calcd 377.09.Tautomer ratio = 1:0.2; yield: 50%; 7Compound 1H NMR  δ 9.98 , 9.40 , 7.47 , 7.38–7.33 , 7.30 , 7.05–7.00 , 6.99 , 4.72 , 3.80 ; 13C NMR  δ 180.3, 174.2, 149.7, 147.3, 137.7, 130.2, 129.1, 128.2, 128.0, 127.1, 126.0, 122.9, 116.0, 112.9, 56.1, 48.1; LRMS (ESI−) m/z 339.45 (M−H)−, calcd 339.08, LRMS (ESI+) m/z 363.35 (M+Na)+, calcd 363.08.Tautomer ratio = 1:0.1; yield: 55%; 8Compound 1H NMR  δ 10.20 , 9.95 , 9.83 , 7.84 , 7.36 , 7.33 , 7.29 , 7.15 , 6.40 , 6.37 , 4.69 ; 13C NMR  δ 180.7, 174.3, 161.1, 159.1, 137.9, 129.6, 129.0, 128.1, 128.0, 125.4, 123.2, 112.9, 108.3, 103.0; LRMS (ESI+) m/z 349.30 (M+H)+, calcd 349.06; HRMS (ESI+) m/z C17H15N2O3S (M+H)+ calcd 327.0798, obsd 327.0794, m/z C17H14N2NaO3S (M+Na)+ calcd 349.0617, obsd 349.0614.Tautomer ratio = 1:0.2; 2-step yield: 38%; 9Compound 1H NMR  δ 9.93 , 9.63 , 9.35 , 7.42 , 7.38–7.33 , 7.29 , 6.96 , 6.90 , 6.83 , 4.71 ; 13C NMR  δ 180.4, 174.2, 148.1, 146.2, 137.8, 130.7, 129.0, 128.2, 128.0, 125,7, 124.8, 123.3, 116.6, 116.4, 48.1; LRMS (ESI−) m/z 324.96 (M−H)−, calcd 325.06, LRMS (ESI+) m/z 349.09 (M+Na)+, calcd 349.06.Tautomer ratio = 1:0.2; yield: 36%; 10Compound 1H NMR  δ 9.92 , 7.80 , 7.40–7.32 , 7.29 , 6.68 , 6.64 , 4.71 , 3.86 , 3.81 ; 13C NMR  δ 179.9, 173.8, 162.2, 159.4, 137.3, 129.2, 128.6, 127.7, 127.5, 125.6, 123.8, 115.3, 106.1, 98.6, 55.9, 55.6, 47.6; LRMS (ESI−) m/z 353.30 (M−H)−, calcd 353.10, LRMS (ESI+) m/z 377.25 (M+Na)+, calcd 377.09.Tautomer ratio = 1:0.2; yield: 58%; 11Compound 1H NMR  δ 9.94 , 7.55 , 7.36–7.24 , 7.12–7.07 , 7.02 , 4.71 , 3.80 ; 13C NMR  δ 180.2, 174.1, 150.5, 149.3, 137.5, 130.2, 128.9, 128.1, 127.9, 127.2, 126.3, 123.2, 113.2, 112.3, 56.0, 55.9, 48.2; LRMS (ESI−) m/z 353.32 (M−H)−, calcd 353.10, LRMS (ESI+) m/z 377.24 (M+Na)+, calcd 377.09.Tautomer ratio = 1:0.2; yield: 52%; 12Compound 1H NMR  δ 9.95 , 9.11 , 7.54 , 7.38–7.32 , 7.29 , 6.87 , 4.71 , 3.80 ; 13C NMR  δ 180.3, 174.1, 148.6, 138.1, 137.8, 130.8, 129.0, 128.1, 128.0, 125.6, 124.7, 107.8, 56.4, 48.1; LRMS (ESI−) m/z 369.33 (M−H)−, calcd 369.09, LRMS (ESI+) m/z 393.26 (M+Na)+, calcd 393.09.Tautomer ratio = 1:0.1; yield: 56%; 13Compound 1H NMR  δ 9.93 , 7.56 , 7.37–7.28 , 7.28 , 4.69 , 1.39 ; 13C NMR  δ 180.4, 174.1, 156.2, 139.8, 137.7, 131.2, 129.0, 128.1, 128.0, 127.2, 125.7, 125.2, 48.1, 35.1, 30.5; LRMS (ESI−) m/z 421.49 (M−H)−, calcd 421.19, LRMS (ESI+) m/z 445.51 (M+Na)+, calcd 445.19.Tautomer ratio = 1:0.1; yield: 39%; 14Compound 1H NMR  δ 10.04 , 7.57 , 7.39–7.27 , 6.89 , 4.72 , 3.81 , 3.70 ; 13C NMR  δ 180.0, 174.1, 153.6, 139.1, 137.6, 130.1, 129.1, 128.2, 128.1, 128.1, 128.0, 107.4, 60.6, 56.4, 48.2; LRMS (ESI−) m/z 383.32 (M−H)−, calcd 383.11, LRMS (ESI+) m/z 407.26 (M+Na)+, calcd 407.10.Tautomer ratio = 1:0.2; yield: 54%; l-Tyrosine and l-dopa were used as substrates to evaluate the enzyme activity. In brief, an aqueous solution of mushroom tyrosinase  was added to each well of a 96-well microplate containing 170 µL of substrate mixture consisting of 345 μM l-tyrosine, 17.2 mM sodium phosphate buffer (pH 6.5), and 10 µL of BABT derivative  in DMSO) or 10 µL of kojic acid. Assay mixtures were incubated at 37 °C for 30 min, and dopachrome amounts synthesized were determined by measuring the optical density of each well at 475 nm using a microplate reader .Mushroom tyrosinase inhibitory activities of BABT derivatives were determined as described previously , with mi7 and 8 were obtained in the presence of l-dopa as the substrate. Briefly, 20 µL aliquots of mushroom tyrosinase aqueous solution (200 units/mL) were added to each well of a 96-well plate containing 10 µL BABT derivative (7 or 8) DMSO solution  and 170 µL of an aqueous solution containing l-dopa  and sodium phosphate buffer . The initial rate of dopachrome formation was determined by examining the changes in absorbance at a wavelength of 475 nm (ΔOD475/min) using a microplate reader .Lineweaver–Burk plots of derivatives 7 and 8) was performed using the Schrödinger Suite (2021-2), as described previously [mTYR) with 2Y9X (Agaricus bisporus) PDB ID was imported from PDB to the Protein Preparation Wizard in Maestro 12.4. Unwanted protein chains in the mTYR crystal structure were deleted. The structure was optimized by deleting water molecules >3 Å from the enzyme and adding hydrogen atoms. The glide grid of the mTYR active site was determined using the binding location of the ligand tropolone, as determined using literature data [7 and 8) were imported to the entry list of Maestro in CDXML format and developed using LigPrep prior to ligand docking. Chemical structures were docked to the tyrosinase glide grid using the Glide task list [An in silico analysis of the chemical interactions between mushroom tyrosinase and the BABT derivatives  complex structures. Tyrosinase molecular force field parameters were written in Gromos53a6 force field format, and 7, 8, or kojic acid molecular force field parameters were derived from Automated Topology Builder  written in Gromos 54a7 force field format, which was converted into Gromacs format data. At the beginning stage, energy minimization was executed using a steep descent method of 50,000 steps to have a stable conformation. After minimization, canonical ensembles (NVT) and isobar isothermal ensembles (NPT) were performed, respectively, with a constant temperature of 300 K for 100 ps for NVT, followed by a constant temperature of 300 K and a constant pressure of 1 atm per 100 ps for NPT. The production MD runs were then performed for 100 ns, keeping the temperature at 300 K and the pressure at 1 bar. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), and hydrogen bonds between tyrosinase and 7, 8, and kojic acid were calculated after the runs. The resulting graphics for these parameters were designed using the Gnuplot program.The Gromacs 2022.2 program was utilized to perform molecular dynamics simulations of the tyrosinase–hTYR) homology model was used for a docking simulation with kojic acid and 8 [8) were docked with the hTYR model using the protocols described above for in silico mTYR docking.A previously created human tyrosinase id and 8 ,23. Comp2 atmosphere.Murine melanoma cells (B16F10) were purchased from the American Type Culture Collection . Streptomycin, Dulbecco’s modified Eagle’s medium (DMEM), penicillin, fetal bovine serum (FBS), trypsin, and phosphate-buffered saline (PBS) were purchased from Gibco/Thermo Fisher Scientific . B16F10 cells were cultured at 37 °C in DMEM containing streptomycin (100 µg/mL), 10% heat-inactivated FBS, and penicillin (100 IU/mL) in a 5% CO4 cells per well and cultured at 37 °C in a 5% CO2 atmosphere for 24 h. The next day, cells were treated with 7 or 8 at six different concentrations  and incubated at 37 °C in a 5% CO2 atmosphere for 72 h. Then, the EZ-Cytox solution  was added to each well. After 2 h of incubation at 37 °C, the optical density of each well was measured at 450 nm using a microplate reader .Cytotoxicity assays in B16F10 cells were performed using the EZ-Cytox solution  . Briefly8 on melanin production was measured using a standard melanin content assay [5 cells per well and allowed to adhere to the well bottoms for 24 h under the conditions used for cell cultures. B16F10 cells were treated with kojic acid  or four different concentrations  of 8 for 1 h and treated with a stimulator (0.5 µM α-MSH plus 200 µM IBMX). After incubation for 72 h at 37 °C in a 5% CO2 atmosphere, melanin levels were determined. To measure the extracellular melanin levels, the absorbance of each well of the culture medium was measured at 405 nm. Intracellular melanin levels were determined as follows: B16F10 cells treated with a stimulator (0.5 µM α-MSH plus 200 µM IBMX) in the presence or absence of test samples (8 and kojic acid) were cultured for 72 h. Cultured cells were washed twice with PBS, and the pellets obtained were dissolved using 1 N NaOH solution  at 60 °C for 1 h. Cell lysates were transferred to the wells of a 96-well plate, and the optical density of melanin dissolved in each well was measured at 405 nm in aqueous DMSO solution using a microplate reader .The effect of nt assay , with mi8 on B16F10 cellular tyrosinase activity was evaluated by measuring the oxidation rate of l-dopa, as previously described [5 cells/well in a 6-well plate and allowed to adhere to well bottoms for 24 h. B16F10 cells were treated with kojic acid at 20 µM or with 8 at four different concentrations  for 1 h. Then, a stimulator (0.5 µM α-MSH plus 200 µM IBMX) was applied to increase the cellular tyrosinase activity, and the cells were cultured for 72 h under the conditions used for cell cultures. Cultured cells were washed twice with PBS and exposed to an aliquot (100 µL) of the lysis buffer solution containing 20% Triton X-100 (5 µL), 50 mM phosphate buffer , and 2 mM phenylmethylsulfonyl fluoride (5 µL) to lyse the cultured cells for 30 min at −80 °C. After defrosting, the cell lysates were transferred to microcentrifuge tubes and centrifuged for 30 min at 4 °C and 12,000 rpm, and the supernatants (80 µL) in each well were mixed with 20 µL l-dopa (2 mg/mL) and incubated at 37 °C for 10 min. The absorbance of dopachrome was measured at 475 nm using a microplate reader .The inhibitory effect of derivative escribed , with mi1–14 were measured. A DPPH (0.2 mM) and methanol solution (180 µL) was mixed with 20 µL of a DMSO solution containing 5 mM BABT derivative, or with 20 µL of an aqueous solution of 5 mM l-ascorbic acid, in each well of a 96-well plate and kept at ambient temperature in the dark for 30 min. The optical density of each well was measured at 517 nm using a microplate reader . l-Ascorbic acid was used as a positive reference control, and the DPPH radical scavenging abilities of BABT derivatives were compared with those of l-ascorbic acid. All experiments were performed independently, thrice.According to previously described methods , with mi1–14 were assessed. Briefly, an ABTS radical solution was prepared by mixing 10 mL of aqueous ABTS solution (7 mM) with 10 mL of potassium persulfate aqueous solution (2.45 mM), and the mixture was kept for 11–15 h in the dark at room temperature until the absorbance remained constant. Prior to the antioxidant assay experiments, the ABTS radical solution was diluted with water to adjust the absorbance at 734 nm to 0.70 ± 0.02. A 10 µL aliquot of BABT derivatives or Trolox at a concentration of 1000 µM was added to 90 µL of ABTS+ free radical solution in each well of a 96-well plate and incubated for 2 min in the dark at ambient temperature. The absorbance of each well was measured at 734 nm using a microplate reader . The ABTS+ radical scavenging activities of BABT derivatives were compared to those of Trolox. All experiments were carried out independently, thrice.According to a previously described method , with miThe ROS scavenging activities of the BABT derivatives were measured using an oxidant-sensitive fluorescent probe ,38. DeriThe peroxynitrite scavenging activities of the BABT derivatives were assessed using the method developed by Kooy , involvi5 cells/mL for 24 h, washed with cold PBS, and harvested. Cytosolic and nuclear fractions of cell lysates were extracted using lysis buffer, as described previously [http://www.attokorea.co.kr, accessed on 21 November 2022).B16F10 cells were seeded in a 60 mm dish at a density of 5 × 10eviously . Proteinp-values < 0.05 were considered to be significant.One-way analysis of variance, followed by a Bonferroni post hoc test, was used to determine the significant differences between treatments. Statistical analysis was conducted using GraphPad Prism 5 . Results are represented as the mean ± standard error of the mean. Two-sided 7 and 8, which showed stronger mushroom tyrosinase inhibition than kojic acid, were proven to be competitive inhibitors of mushroom tyrosinase in the kinetic study using Lineweaver–Burk plots. In silico docking simulation results using mushroom tyrosinase indicated that they bound to the active site of tyrosinase, supporting the kinetic study results. In experiments using B16F10 cells, 8 exhibited stronger inhibition of melanin production and cellular tyrosinase activity than did kojic acid, and the similar pattern of inhibition of tyrosinase activity and melanin production indicated that the anti-melanogenic effect of 8 was due to tyrosinase inhibition. In addition, 8 potently removed the DPPH and ABTS radicals and peroxynitrite and suppressed melanogenesis-associated protein expression, which indicates its potential as an attractive anti-melanogenic agent. These results suggest that derivative 8 may be used as an effective multifunctional tyrosinase inhibitor.To identify a new tyrosinase inhibitor, we designed BABT derivatives based on the structure of MHY2081 in this study. Of the 14 BABT derivatives tested, two exhibited strong inhibitory activities against mushroom tyrosinase. BABT derivatives


Dairy foods are essential in the diet, although in some susceptible individuals they may cause allergy to cow’s milk proteins. Therefore, alternative methods are sought to reduce their allergenicity. Transglutaminase (TG) is widely used in dairy products mainly to improve texture. Although it has been claimed that TG can be used to modify the digestibility and allergenicity of foods, its impact within a real matrix has been rarely studied. The aim of this work was to assess the allergenic potential of crosslinked skim milk (SM), milk casein fraction (CN), and whey protein (WP). To this purpose, inhibition ELISA with sera from milk allergic patients, in vitro activation tests of mouse mast cells and splenocytes, and simulated gastrointestinal digestion assays were performed. The results showed that cross-linking increased the binding of IgE to WP, but decreased IgE-binding to SM and CN. However, no differences were observed in the ability of cross-linked proteins to induce mast cell degranulation compared to native proteins. The cross-linking of SM and CN reduced Th2 cytokine release from the splenocytes of sensitized mice. All TG-treated samples exhibited more resistance to in vitro digestion than the untreated proteins and the human IgE binding capacity after digestion was higher. In conclusion, TG treatment of milk proteins does not reduce the risk of eliciting allergic symptoms in cow’s milk allergic patients. Food allergies are increasing around the world with unprecedented complexity and severity. They constitute a serious health problem that causes large public health costs and severely deteriorates the quality of life of patients. Of all known food allergies, cow’s milk allergy is of special interest due to its high incidence during the first three years of life . MainstaDifferent approaches have been used to try to produce hypoallergenic foods, taking advantage of the transformations that food components undergo during processing . Among tPartial hydrolysis of whey proteins or casein followed by TG-catalyzed crosslinking of the hydrolysates was proposed as a strategy to produce protein polymers with lower antibody binding properties but with improved textural and sensory properties ,7. RegarThe aim of this work was to assess the effect of TG-mediated crosslinking on the IgE-binding, ability to induce mast cell degranulation, and immunostimulating properties of milk in comparison with its two main protein fractions. Moreover, a physiologically relevant in vitro digestion model was used to investigate the susceptibility to digestion of the cross-linked milk proteins and the binding of human IgE to the resulting digests.g at 4 °C for 30 min, filtered through glass wool, lyophilized, and stored at −20 °C. Commercial micellar casein Prodiet 85B (CN) was from Ingredia S.A, . Whey protein isolate (WP) (LACPRODAN DI-9224K) was from Arla Foods Ingredients . Protein concentration of skim milk (SM), CN, and WP was determined by the Kjeldahl method [®-MP, Ajinomoto Co., Sao Paulo, Brazil; enzymatic activity:100 U/g), a commercial powder including 1% TG and 99% maltodextrin. The enzyme was dissolved in PBS and mixed  with the SM, CN, and WP solutions to achieve a final protein concentration of 2.5 mg/mL and a final enzyme activity of 1, 10, 100 and 1000 U in the reaction mixture. TG cross-linking experiments were carried out at 40 °C for 1, 3, 8, and 24 h. Then, samples were heated at 90 °C for 10 min for enzyme inactivation.Milk, obtained from the local farm La Colmenareña , was skimmed by centrifugation, 3000× l method . The absTM cuvette on preform bis–tris gels (12%) at 120 V, using XT-MES as running buffer. Samples were diluted in sample buffer with 5% (v/v) β-mercaptoethanol to a final protein concentration of 250 µg/mL, then heated at 95 °C for 10 min. Hence, 30 μL of sample were placed into each lane. Staining was completed with Bio-Safe Coomassie G-250. All equipment and reagents were from Bio-Rad .Electrophoresis was performed in a CriterionHuman IgE-binding to native and TG cross-linked SM, CN, and WP and their digests was evaluated by inhibition ELISA , using a6 cells) were sensitized with serum from BALB/c mice orally sensitized with 5 mg of SM, CN, and WP plus 10 μg of cholera toxin  [Bone marrow-derived mast cells (BM-MCs) were in vitro differentiated into a mucosal-like phenotype as described previously . BrieflyCA, USA) . After wCA, USA) . The perThe cell viability assay was carried under the culture conditions described below and determined by 3--2,5-diphenyltetrazolium bromide (MTT) method. Hence, 20 μL of MTT solution (5 mg/mL PBS) were added to each well. Samples were incubated for a further 4 h in dark. Then, media were removed, and 100 μL of dimethyl sulfoxide:methanol (1:1) was added to dissolve the purple insoluble MTT formazan, and the absorbance was measured at 570 nm in a Fluorostar Optima microplate-reader  using a 650 nm filter as a reference.n = 5). After sacrifice, whole spleens were excised, and cell suspensions were prepared. Splenocytes were cultured in RPMI 1640 plus 10% of fetal bovine serum, L-glutamine (2 mM), penicillin (50 U/mL) and streptomycin (50 μg/mL)  at 4 × 106 cells/mL in 48-well plates. Stimuli consisted of 200 μg/mL of untreated or TG cross-linked SM, CN, WP, and PBS as negative control. Control and stimulated splenocyte cultures were maintained for 72 h at 37 °C in a 5% CO2 gassing incubator and were performed in duplicate. The supernatants were collected and kept frozen (−80 °C) until assayed for IL-4 and IL-13 quantification using mouse ELISA kits .Splenocytes were obtained from female BALB/c mice orally sensitized with SM, CN, and WP (w/w), and incubating for 30 min at 37 °C. Stopping of the reaction was achieved by heating at 95 °C for 10 min.Simulated gastrointestinal digestion of native and TG cross-linked SM, CN, and WP was performed as described previously . Brieflyv/v) trifluoroacetic acid from Scharlau Chemie  in double-distilled water as phase A, and 0.27% (v/v) trifluoroacetic acid in HPLC-grade acetonitrile  as phase B. The gradient was linear, increasing B in A from 0% to 60% at a flow rate of 1 mL/min during 90 min, followed by a 35-min wash with 60% and 10 min with 100% of solvent B. Detection was at 214 nm and data were processed by using Empower 2 Software from Waters.RP-HPLC analysis was conducted on a Waters 600 HPLC instrument  with a RP318 column . 100 µL (2 mg/mL) of sample were injected and eluted for 60 min using 0.37% . Data are expressed as the mean ± SEM of 3 independent experiments.Statistical analyses were performed using GraphPad Prism software, version 7.0e (GraphPad). The two-tailed Student’s S1- and β-CNs, which allow a greater exposure of reactive groups and an increased accessibility to the enzyme [CN, WP, and SM were susceptible to TG mediated cross-linking . As expee enzyme . The pree enzyme . Monomere enzyme . Althouge enzyme ,20. We eIgE reactivity of pooled sera from milk allergic patients from our serum library  towards The contribution of individual milk proteins to the binding of IgE capacity to SM, CN, and WP was assessed by inhibition ELISA pre-coating the plates with either β-CN, β-Lg, or α-la. In SM, the binding of IgE to β-CN was greatly reduced after treatment with TG to 3% of the IgE binding to β-CN in the untreated sample b. HoweveBecause mast cells are the main effector cells of the allergic response, we assessed the ability of TG-treated SM, CN, and WP to activate them through an IgE dependent pathway using BM-MCs . Cells wThe immunostimulating properties of TG-treated SM, CN, and WP were evaluated in splenocytes from SM, CN, and WP sensitized BALB/c mice, respectively . StimulaSome studies have determined the influence of crosslinking on the allergenic properties of proteins through the analysis of the IgE binding capacity by ELISA, Western blotting, or degranulation tests using basophil and mast cell cultures ,31,32, bNone of the samples analyzed in this study exhibited spleen cell cytotoxicity after a 72 h-culture , indicatThe resistance to in vitro gastric digestion of TG treated SM, CM, and WP was higher than that of the native proteins, and this was especially noticeable during the first minutes of pepsin treatment . ResistaThe peptide profile generated during digestion in the gastric and duodenal phases was similar in the native samples and in those treated with TG. Similar results were found by Fotschki et al., who observed that TG influenced the gastric digestibility of cow and horse milk, slowing digestion of the proteins . Cross-lAs expected, in vitro gastrointestinal digestion significantly reduced the IgE-binding capacity of SM, CN, and WP . The conTG was effective in crosslinking SM, CN, and WP, forming high molecular weight polymers, although a substantial amount of unmodified monomeric serum proteins remained. Although the binding of human IgE to β-CN was significantly reduced and that to β-LG and α-LA was increased after treatment of SM, CN, and WP with TG, no differences were observed in either case in the ability to induce mast cell degranulation compared to the untreated proteins. SM and CN subjected to cross-linking induced a lower release of Th2 cytokines from splenocytes of mice sensitized, respectively, to SM and CN, but WP treated with TG did not modify the production of IL-4 and IL-13 with respect to the native protein in spleen cells from WP-sensitized mice. TG-treated SM, CM, and WP were more resistant to digestion than the untreated proteins and, consequently, the binding of human IgE to their digests was also higher. These results question the use of crosslinking with transglutaminase to obtain hypoallergenic products.


Dendrobium catenatum is a widely cultivated Chinese orchid herb rich in abundant secondary metabolites, such as terpenes. However, terpene distribution and characterization of terpene biosynthesis-related genes remain unknown in D. catenatum. In this study, metabolic profiling was performed to analyze terpene distribution in the root, stem, leaf, and flower of D. catenatum. A total of 74 terpene compounds were identified and classified. Clustering analysis revealed that terpene compounds exhibited a tissue-specific accumulation, including monoterpenes in the flowers, sesquiterpenes in the stems, and triterpenes in the roots. Transcriptome analysis revealed that the ‘terpenoid backbone biosynthesis’ pathway was only significantly enriched in root vs. flower. The expression of terpene biosynthesis-related genes was spatiotemporal in the flowers. Prenylsynthase-terpene synthases (PS-TPSs) are the largest and core enzymes for generating terpene diversity. By systematic sequence analysis of six species, 318 PS-TPSs were classified into 10 groups and 51 DcaPS-TPSs were found in eight of them. Eighteen DcaPS-TPSs were regulated by circadian rhythm under drought stress. Most of the DcaPS-TPSs were influenced by cold stress and fungi infection. The cis-element of the majority of the DcaPS-TPS promoters was related to abiotic stress and plant development. Methyl jasmonate levels were significantly associated with DcaTPSs expression and terpene biosynthesis. These results provide insight into further functional investigation of DcaPS-TPSs and the regulation of terpene biosynthesis in Dendrobium. Terpenes are the largest and most diverse class of chemical substances that have been found in the plant kingdom, from moss to angiosperm . Plants TPS genes in seven species ranges from 1 in Physcomitrella patens to 69 in Vitis vinifera [AtTPS genes have distinct tissue-specificity and response mechanisms, such as AtTPS21 in the stigma and sepals and AtTPS03 in pollen [AtTPS02 and AtTPS03 are induced locally at wound sites [Plant PSs are a class of enzymes responsible for the synthesis of isoprenoids, which contain three enzymes: geranylgeranyl pyrophosphate synthase (GGPS), farnesyl pyrophosphate synthase (FPS), and geranyl pyrophosphate synthase (GPS) . The PSsvinifera . In Arabvinifera . AtTPS gn pollen , and AtTD. catenatum  belongs to the Orchidaceae family and is a rare and precious Chinese herb. D. catenatum contains many bioactive extracts including polysaccharides, alkaloids, phenols, terpenes, coumarins, and flavonoids [Flavanone 3-hydroxylase (F3H) and leucoanthocyanidin dioxygenase (LDOX) affected the flux of dihydroflavonol and led to flavonoid accumulation in the stem [D. catenatum. In recent years, D. catenatum has been successfully cultivated and has become an important economic herb in China. Due to its antioxidant and antitumor activities, demand for secondary metabolites of D. catenatum is increasing across medicinal and therapeutic applications. To obtain a research basis for terpene metabolism in Dendrobium plants, we first identified the PS-TPS family in the D. catenatum genome and then made comprehensive assessments of the phylogenetic relationship, motif distribution, gene-specific expression, and responses to abiotic and biotic stresses. Our study provides insights into the evolution and function of PS-TPSs in medicinal Dendrobium plants.avonoids . The genavonoids . Flavanothe stem . HoweverD. catenatum  was performed on transcriptome data from four tissues. The KEGG pathway ‘terpenoid backbone biosynthesis’ was only significantly (p < 0.05) enriched in root vs. flower (Dca010855) was highly expressed in the stem. Two genes, TPS (Dca026890) and SPS , were predominantly expressed in the leaf. We further explored the expression of terpene biosynthesis-related genes in early flower buds (F1), medium flower buds (F2), and bloomed flowers (F3). The GSEA showed that ‘terpenoid backbone biosynthesis’ was only significantly (p < 0.05) enriched in F3 vs. F1 , and 247 TPSs were divided into seven groups  according to previous studies D (‘x’ is any amino acid) motif and CxxxC motif were found in all DcaPSs, except DcaFPS (2–4)D motif, while the GGPS small subunit (SSU) lacked this motif and contained the CxxxC motif [2–4)D and CxxxC motifs  synthases [S. moellendorffi containing only the ‘terpene_synth_C’ domain represented a type of TPS that has been widely identified in bacteria, fungi, and nonseed plants [D. catenatum.Furthermore, we searched for 30 conserved motifs in DcaTPSs using MEME software. Most subfamily members showed similar conserved motif distributions . Howeverynthases . The phyd plants . Thus, DDcaPS-TPSs during growth and development, RNA-seq data from different plant tissues and organs were detected [DcaPS-TPSs showed distinct organ-specific expression patterns, and the half of DcaPS-TPSs were primarily expressed in flowers (DcaTPS25 and -03 in lip (labellum), DcaGGPS4 and DcaTPS23 in flower bud, and DcaTPS17 and -18 in sepal. Furthermore, we explored the expression levels of DcaPS-TPSs during different flowering phases. DcaSPS, DcaGPS2, DcaGGPS1, and most DcaTPSs were highly expressed in F3. The rest of the DcaPS-TPSs were primarily expressed in F1/F2 , freezing treatment (FT), and post-freezing recovery (FR). Fourteen DcaPS-TPSs were downregulated, and six DcaPS-TPSs were significantly upregulated under cold stress. The expressions of three DcaPS-TPSs were only induced under FT including DcaGGPS2 and DcaTPS23 and -30 , plant growth and development-related cis-elements , and phytohormone-related cis-elements . Most of the DcaPS-TPS promoters contained light-responsive elements, suggesting that DcaPS-TPSs might play important roles in plant photoperiod regulation.To predict the putative functions of aPS-TPSs a. PotentDcaPS-TPS promoters contained CGTCA-motifs of JA response  in Pr vs. CK  not only steer the response of plant stress but also induce the accumulation of secondary metabolite production ,19. Mostresponse . Our prein stems . The endr vs. CK . We selereatment d. These D. catenatum displays strong immune modulatory activity, and is used for relieving stomach upsets and its antitumor and antipyresis properties [D. catenatum primarily focuses on phenylpropanoid biosynthesis such as flavonol and anthocyanin [Dendrobium [Dendrobium stem [D. catenatum. In this study, 41 volatile terpenes and 33 non-volatile terpenes were detected D pyrophosphate-binding motifs . The genome of Dendrobium catenatum was downloaded from NCBI . The PS-TPS proteins were originally obtained using the hidden markov model (HMM) search and BLASTP search. Three domains, PF03936, PF19086 and PF01397 were used to search for TPSs, and PF00348 was used to search for PSs. The significant hits were selected with an E value < 1 × 10 −10 as the cutoff. All the hits were further confirmed on NCBI-CDD  and SMART .The PS-TPS sequences including genomic DNA and proteins in https://meme-suite.org/meme/, accessed on 2 April 2021). The cis-elements of DcaPS-TPSs promoters were analysed in PlantCARE .The sequences of PS-TPS proteins from these species were aligned with ClustalW software and then an un-rooted tree was constructed using FastTree with options by default ,40. The DcaPS-TPSs, the raw RNA-seq data of tissues (PRJNA348403 and PRJNA680456) were downloaded from the NCBI [−2 s−1; 28/22 °C day/night; and relative humidity 60/70% day/night) and adapted to the controlled conditions for 10 days. Plants were irrigated on the 1st day, dried from the 2nd to the 7th day, and then resumed on the 8th day. The raw RNA-seq data of drought stress were obtained from Sci Data [C. gloeosporioides for 25 d in greenhouse . The raw RNA-seq data of C. gloeosporioides infected plants were submitted to the BIG Data Center of the Chinese Academy of Sciences (https://bigd.big.ac.cn (accessed on 6 May 2022)) with accession number CRA002691. For cold stress, two-year-old cultivated plants were placed at 0 °C and then subjected to a gradual drop from 0 to −6 °C within 3 h. The temperature was held at −6 °C for 3 h and then held at 8 °C for 24 h without lighting. The raw RNA-seq data of cold stress have been submitted to the BIG Data Center with accession number CRA003229 and CRA005177 [DcaPS-TPSs expression matrices were treated by hierarchical clustering using the R package ggplot2 .For expression profiling of the NCBI ,41. For Sci Data . The RNASci Data . For funRA005177 . The quaRA005177 . In brieD. catenatum were grown at 20 ± 2 °C with a light/dark cycle of 12/12 h and a 65–75% relative humidity as a control treatment. For MeJA treatment, the leaves were sprayed with 1 mM MeJA for 24 h. The leaves were harvested and immediately frozen in liquid nitrogen.Two-year-old cultivated Total RNA was isolated from leaf samples using TransZol reagent . First-strand cDNA was reverse transcribed using the TIANscript RT Kit according to the manufacturer’s instructions . Quantitative real-time PCR analyses were performed using a SYBRGreen qPCR kit (TransGenBiotech) with a MyiQ system  as described previously . The priD. catenatum were grown in greenhouse . The D. catenatum flowers were in full bloom in May. The tissue samples were collected from six independent plants  and immediately ground into powder in liquid nitrogen.Two-year-old cultivated m/z. The quadrupole mass detector, ion source and transfer line temperatures were set at 150, 230, 280 °C, respectively. Compounds were identified by matching the data from the MWGC library.For volatile organic compounds analysis, 1 g powder was transferred to an SPME vial containing 10 μL heptanone as an internal standard. A preconditioned SPME fiber  was then exposed to the headspace of the capped vial at 100 °C for 15 min. The fiber coating was carried out in the injection chamber of the GC (Agilent 8890) in splitless mode at 250 °C for 5 min. Quantification of volatiles was carried out using GC (Agilent 8890) and mass spectrometer (Agilent 5977B), and equipped with a capillary DB-5MS column (30 m × 0.25 mm inner diameter with 0.25 μm film thickness). Helium was used as the carrier gas at a linear velocity of 1.0 mL/min. The injector temperature was kept at 250 °C and the detector at 280 °C. The GC oven temperature was programmed at 40 °C for 2min, increased from 40 to 280 °C at 7 °C/min, and then held for 5 min. The mass spectra were taken at electron ionization at 70 eV, and the mass range was 50–500 For non-volatile organic compounds analysis, 100 mg powder was dissolved in 1.2 mL 70% methanol solution at 4 °C overnight. UPLC-ESI-MS/MS conditions were performed according to our previous work . The met


Phosphorylation is an essential process in biological events and is considered critical for biological functions. In tissues, protein phosphorylation mainly occurs on tyrosine (Tyr), serine (Ser) and threonine (Thr) residues. The balance between phosphorylation and dephosphorylation is under the control of two super enzyme families, protein kinases (PKs) and protein phosphatases (PPs), respectively. Although there are many selective and effective drugs targeting phosphokinases, developing drugs targeting phosphatases is challenging. PTP1B, one of the most central protein tyrosine phosphatases (PTPs), is a key player in several human diseases and disorders, such as diabetes, obesity, and hematopoietic malignancies, through modulation of different signaling pathways. However, due to high conservation among PTPs, most PTP1B inhibitors lack specificity, raising the need to develop new strategies targeting this enzyme. In this mini-review, we summarize three classes of PTP1B inhibitors with different mechanisms: (1) targeting multiple aryl-phosphorylation sites including the catalytic site of PTP1B; (2) targeting allosteric sites of PTP1B; (3) targeting specific mRNA sequence of PTP1B. All three types of PTP1B inhibitors present good specificity over other PTPs and are promising for the development of efficient small molecules targeting this enzyme. PTP1B is a ubiquitous and abundant intracellular prototypic non-receptor tyrosine phosphatase that is recognized as a major negative regulator of numerous signaling cascades of metabolic and/or oncogenic relevance such as the insulin or epidermal growth factor (EGF) pathways ,3. In thAs stated above, given its key functions in several signaling cascades, PTP1B activity is regulated by several mechanisms that may work in tandem to modulate the enzyme’s functions. Especially, reversible and irreversible post-translational modifications (PTMs) such as phosphorylation, oxidation, sumoylation, and proteolysis, were put forward as essential for regulating PTP1B activity ,7,8,9,10PTP1B is one of the major non-receptor protein tyrosine phosphatases (PTPs) whose first crystal structure was solved in 1994 . At the The N-terminal catalytic domain of PTP1B is regulated through the PTMs of key residues found within this domain: (i) phosphorylation of crucial serine (Ser) and tyrosine (Tyr) residues, and (ii) oxidation of the catalytic cysteine (Cys) 215 in response to reactive oxygen species (ROS).66YINA69, is consistent with the consensus binding site (YXNX) for the Src homology 2 (SH2) domain of Grb2 (growth factor receptor-related protein 2)  benzoic acids PTP1B inhibitors were developed and showed excellent inhibition efficiency ,104,105.50 is less than 15 µM), their specificity towards other PTPs, especially TCPTP, is unknown.This site is located between the β-sheet comprising residues Leu71 and Lys73 and a lipophilic pocket involving residues Arg79, Pro206 to Pro210  Figure E. CompouASOs generally refers to a short-stranded nucleic acid (about 15-25 nucleotides) that has undergone some chemical modifications. Its base sequence is complementary to the specific sequence of the target mRNA. After entering the cells, it can form a double-stranded structure with the target mRNA sequence, blocking protein synthesis, and finally stopping the translation machinery . Until nTo date, only two ASOs inhibitors targeting PTP1B have been designed by Ionis Pharmaceuticals, Inc. (USA): IONIS 113715 and IONIS PTP1BRx . IONIS 12+, Fe2+, Cu2+, Cd2+, Mg2+) result in the loss of PTP1B phosphatase activity [Over the past few decades, around 500 PTP1B inhibitors  have been identified/developed. They mostly interfere with the catalytic active site of PTP1B . For exaactivity ,115. HowSpecificity is always a hurdle in drug discovery, especially for phosphatase inhibitors. The process of discovering PTP1B inhibitors has been a long journey for researchers over the past decades due to two challenges.The first challenge is selectivity. The discovered PTP1B inhibitors act primarily on the catalytic site of the enzyme. However, the catalytic site of non-receptor PTPs is highly conserved, in particular TCPTP which shares 72% sequence identity and 86% similarity in the catalytic domain with PTP1B , and thuThe second challenge is cellular permeability and bioavailability. The catalytic site of PTP1B is positively charged, so inhibitors acting on the catalytic site are ideally negatively charged or highly polar. However, negatively charged or highly polar inhibitors cannot effectively cross the cell membrane, and thus reach the phosphatase. Different approaches  and dephosphorylation (by protein phosphatases) play an essential role in cellular regulation and signal transduction. Although there are many specific and effective drugs on the market targeting kinases, the development of drugs targeting phosphatases has proven to be challenging. As stated above, some inhibitors have shown an excellent inhibitory effect on PTP1B, but their study was discontinued because of their low specificity and side effects. In this mini-review, three categories of PTP1B inhibitors were reviewed with the aim of informing the inhibition mechanisms of specific PTP1B inhibitors. The cellular permeability and bioavailability of these inhibitors are still poorly understood. Therefore, other related tests, such as pharmacokinetic properties, should be further investigated.


Although ECP production is attributed to eosinophils, we reported that ECP is also present in neutrophils from allergic patients where, in contrast to eosinophils, it is produced in an IgE-dependent manner. Given the key role of ECP in asthma, we investigated the molecular mechanisms involved in ECP production as well as the effects induced by agonists and widely used clinical approaches. We also analyzed the correlation between ECP production and lung function.Neutrophils are involved in the pathophysiology of allergic asthma, where the Eosinophil Cationic Protein Neutrophils from allergic asthmatic patients were challenged with allergens, alone or in combination with cytokines, in the presence of cell-signaling inhibitors and clinical drugs. We analyzed ECP levels by ELISA and confocal microscopy. Lung function was assessed by spirometry.IgE-mediated ECP release is dependent on phosphoinositide 3-kinase, the extracellular signal-regulated kinase (ERK1/2) and the production of reactive oxygen species by NADPH-oxidase. Calcineurin phosphatase and the transcription factor NFAT are also involved. ECP release is enhanced by the cytokines interleukin (IL)-5 and granulocyte macrophage-colony stimulating factor, and inhibited by interferon-γ, IL-10, clinical drugs  and allergen-specific IT. We also found an inverse correlation between asthma severity and ECP levels.in vitro ECP production by peripheral neutrophils.Our results suggest the molecular pathways involved in ECP production and potential therapeutic targets. We also provide a new method to evaluate disease severity in asthmatic patients based on the quantification of  It is involved in host defense against parasites, bacteria and viruses, and regulates immune and epithelial cell function, complement, coagulation and fibrinolysis . ECP was airways . ECP has airways , 8, indiNeutrophils are the first cells to reach the inflammation foci in the asthmatic airways, where they perform inflammatory functions . PatientThe expression of IgE receptors has been reported in neutrophils –14. We pThe Hospital Universitario Virgen Macarena ethics committee approved the study and each sample donor gave written informed consent.1 (Dermatophagoides pteronyssinus), G3 (Dactylis glomerata), T9 (Olea europaea), M6 and W6 (Artemisia vulgaris) and were purchased from Diater, . Wortmannin, PD098059, SB203580, 4-hydroxy-3-methoxyaceto-phenone (HMAP), 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), cyclosporin A (CsA), cell permeable NFAT inhibitor (VIVIT), tiotropium bromide, budesonide, formoterol, were from Sigma-Aldrich Co . IL-4, IL-5, GM-CSF, IFN-γ, IL-10 were from Preprotech . Ficoll-hypaque, phosphate-buffered saline, RPMI 1640, fetal bovine serum, penicillin/streptomycin and goat anti-human IgE (α-IgE) were purchased from Thermo-Fisher Invitrogen . All cultured reagents had endotoxin levels of ≤ 0.01 ng/ml, as tested by the Limulus lysate assay .The Ags, available as commercial extracts, included Din vitro cellular challenge, oral bronchodilators for 24 h or antihistamines, oral corticosteroids, disodium cromoglycate, or nedocromil sodium in the previous week. The healthy group had no history of allergy or bronchial symptoms, and gave negative skin-prick tests and had specific-IgE to the battery of inhalant Ags. None of the participants in this study suffered infection by SARS-CoV-2 in the month previous to the blood extraction.The study included three groups of adult donors: atopic patients with bronchial asthma  with no 6 cells/300 μl) were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/ml penicillim, and 100 μg/ml streptomycin and maintained in an atmosphere of 95% O2 and 5% CO2. For the stimulation treatments, neutrophils were incubated with 10 μg/ml Ags or α-IgE antibodies at 37°C for the indicated times. Wortmannin, PD098059, SB203580, HMAP, AEBSF, CsA, VIVIT, IL-4, IL-5, IFN-γ, IL-10, GM-CSF, tiotropium, budesonide, and formoterol were added 1 h prior to stimulation and they were previously tested for the optimal concentration without affecting cell viability. In this case, cell health was assayed using the AlamarBlue kit (Thermo-Fisher) according to the manufacturer’s instructions. This kit quantifies the natural reducing power of living cells to convert resazurin to fluorescent resorufin.Highly purified human peripheral blood neutrophils were isolated using the neutrophil isolation kit  following the manufacturer’s instructions. The purity of neutrophils was on average >99% . Cells  according to the manufacturer’s instructions.FEV1 was measured using a spirometer . The best value of three maneuvers was expressed as the percentage of the predicted value. The entire procedure was based on the guidelines of the American Thoracic Society of Standardization .Sputum was induced and processed as previously described . Brieflypost-hoc Tukey’s honest significance tests. Correlation was measured using the Pearson correlation coefficient. Data are expressed as mean ± SEM. A value of p<0.05 was considered significant.All statistical analyses were performed using GraphPad Prism 4.00 for Windows  and R-4.2.1. Multi-group analysis was performed using two-way ANOVA, followed by In previous work, we reported that the crosslinking between Ags/α-IgE and IgE molecules bound to surface IgE receptors  induces the synthesis and release of ECP by human neutrophils from allergic asthmatic patients . This prDuring the first steps of neutrophil activation there is an increase in phosphoinositides through PI3K activation , as wellIn addition, we have shown that the calcineurin phosphatase (CN)/NFAT transcription factor signaling pathway, a key player in neutrophil activation during the immune responses , is alsoIn this context, we aimed to explore the specific role of all these molecular intermediates in ECP production. To this end, we tested the effects of the following protein-specific inhibitors: wortmannin (PI3K inhibitor ), PD0980-16) and stimulation with Ags/α-IgE  both significantly affected neutrophil ECP release and there was a significant interaction between these factors . Tukey’s post-hoc tests showed that wortmannin  (Neutrophil ECP production is modulated by agonists such as or (PAF) . To studor (PAF) , 35, andor (PAF)  and IL-1or (PAF) , that inNeutrophils from allergic patients were preincubated with these cytokines 1h prior the addition of Ags/α-IgE. We then quantified ECP production in the culture supernatant. IL-4 did not modify the basal nor the IgE-dependent ECP production. The same dose of IL-5 did not have an effect on its own, but it significantly enhanced the Ags/α-IgE stimulating effect. Interestingly, GM-CSF displayed a robust response in Ags/α-IgE-treated cells, and was the only cytokine producing significant ECP release in unstimulated cells. On the other hand, IFN-γ and IL-10 significantly reduced ECP release in both stimulated and unstimulated cells Table 2.Long-Acting Beta2-Agonists, LABAs)  (Glucorticosteroids (GC) , broncho, LABAs) , and Lon, LAMAs)  are effe-16) and stimulation  both significantly affected neutrophil ECP release and there was a significant interaction between these factors . Tukey’s post-hoc tests showed that all three drugs, alone or in combination, led to a significant reduction in ECP release, compared to untreated cells, for both Ags and α-IgE stimulation. Considering the drugs alone, Budesonide led to the largest change, followed by formoterol and tiotropium  . Thus, wThe combination of formoterol and tiotropium enhanced the inhibitory effect that they produced alone. This inhibition was more significant when formoterol or tiotropium were combined with budesonide. The mix with all three drugs showed the largest decrease in ECP release Figure 2-16) and Ags/α-IgE stimulation  significantly affected ECP release and there was an interaction between factors . Further analysis using Tukey’s post-hoc tests showed that the group of IT-treated allergic patients experienced a significant reduction in ECP release in the presence of Ags/α-IgE, compared with the non-IT treated patients . Although there was no significant difference in ECP release between IT-treated patients and healthy donors, values were higher in the first group with respect to the second . Note that in healthy donors, neither Ags nor α-IgE led to significant differences in ECP release compared with unstimulated cells, although α-IgE treatment did suggest a possible increase.Ag-specific IT provides long-term reduction in both allergic symptoms and disease progression . This imRegarding IL-5 and GM-CSF, we found that both cytokines induced a significant ~2-3 fold increase in Ags/α-IgE-induced ECP production for both non-IT and IT-treated allergic patients. However, ECP release was significantly lower (p<0.001) in patients who received IT treatment compared to patients who did not    Figure 3+ cells are not always correlated with eosinophil counts  can behave differently . ECP has been immunohistochemically detected not only in eosinophil granulocytes, but also in neutrophils . We coulferently . Therefode novo, and release ECP in response to Ags/α-IgE IgE-receptor crosslinking  and M3-muscarinic receptors (inactivated by LAMAs) are also expressed by neutrophils  and was supported by grants from the Instituto de Salud Salud Carlos III  and Fundació n Alergol, Spain. AV-R was supported by a grant from the Ministerio de Economía y Competitividad  and holds a Nicolás Monardes contract from the Andalusian Health Service (C-0060-2018); PC is under a senior postdoc contract from the Ministry of Health and Families . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.We would like to thank Katherina García and Jose Maria Urbano for their confocal microscopy assistance.RP and DR were employed by Diater.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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The aim of the study is to observe the difference in progression between type 1 and type 2 Bietti crystalline dystrophy (BCD) using multimodal imaging.  A retrospective clinical study was performed with six BCD patients who underwent multimodal imaging twice in Hebei Provincial Eye Hospital from October 2015 to December 2020. Multimodal imaging includes color fundus photography, fundus autofluorescence (AF), infrared autofluorescence (IRAF), fundus fluorescein angiography (FFA), and spectral domain optical coherence tomography (SD-OCT). The fundus lesion progression difference was observed in 3 patients with type 1 BCD and 3 patients with type 2 BCD.  In type 1 BCD, the range of hypoautofluorescence (hypo-AF), hypoinfrared autofluorescence (hypo-IRAF), and hypofluorescence in the posterior pole was enlarged, and FFA showed that the lesions in the posterior pole and periphery extended to the middle periphery. SD-OCT revealed retinal and choroidal thinning, progressive loss of the outer nuclear layer and ellipsoid zone, and reduction of the choroid macrovascular diameter. In type 2 BCD, the range of hypo-AF was enlarged, but there was no significant change in the macula area. The uniform hypo-IRAF in the posterior pole showed no significant change. FFA showed no significant change with the progression of the disease in the macula area and the hypofluorescence around it expanded. SD-OCT revealed no obvious change in the macula area.  The retinal choroid atrophy in the macula area of type 1 BCD continued to worsen, and the choroid great vessels became narrower. The macular lesions of type 2 BCD can remain unchanged for a long time. Bietti crystalline dystrophy (BCD) was first reported by Bietti et al. in 1937 . CurrentThe implementation of all research methods in this study followed the provisions of the Helsinki Declaration, the Ethics Committee of Beijing Tongren Hospital and the Ethics Committee of Hebei Provincial Eye Hospital. This study included 6 BCD subjects who underwent fundus color photography, autofluorescence (AF), infrared autofluorescence (IRAF), FFA, and spectral domain optical coherence tomography (SD-OCT) twice in Hebei Provincial Eye Hospital from October 2015 to December 2020. The clinical features and imaging changes of these patients were retrospectively analyzed.Written informed consent was obtained from each patient before peripheral venous blood was drawn for genomic DNA extraction and mutation screening of the CYP4V2 gene by direct sequencing. The 6 subjects were unrelated Chinese patients, and all diagnosed with BCD by genetic testing.For convenience of description, the 6 patients are referred to as P1, P2, P3, P4, P5, and P6. The interval between the two multimodal imaging was 1 year for P1 and P2, 3 years for P3, and 2 years for P4–P6.Our previous study divided BCD into two types based on the observation of FFA progression in 12 patients with BCD . In typeAll patients underwent a complete ophthalmic examination, including the best corrected visual acuity (BCVA), slit lamp microscopy, Goldman flattening tonometry dilated fundus examination, and fundus photography . AF, IRAF, and FFA images were obtained with a confocal scanning laser ophthalmoscope  with a 55 × 55° field lens.SD-OCT images were obtained by spectral-domain optical coherence tomography . Central macular thickness (CMT) and subfoveal choroid thickness (SCT) were measured in the B-scan flat through the macula fovea. Two large choroid vessels that easily measured were selected for measurement of the vessel diameter, one at the nasal side and one at the temporal side of the choroid below the fovea. The difference of vessels' diameter between the first and last examination was calculated, and the change of vessels' diameter was calculated. The ellipsoid zone (EZ) of the fovea in the right eye of P1 was missing, but the remaining EZ was visible in the temporal side of the fovea. Therefore, the distance between the Bruch membrane of the fovea and the residual EZ of the temporal side was measured, and the difference between the first and last measured values was calculated . A smallThe age of six patients ranged from 30 to 46 years at initial diagnosis. The BCVA of the type 1  and type 2  BCD patients decreased significantly. The genetic data of the patients are shown in With the progression of the disease, the crystalline deposits in the posterior pole fundus of patients with type 1 BCD decreased significantly Figures . In typeThe AF and IRAF of both eyes of the BCD patients were basically the same. The range of hypo-AF in P1–P6 expanded. The hypo-AF region is formed by the combination of multiple small patches of hypo-AF Figures . In typeThe IRAF images of type 1 patients showed hyper-IRAF spots at the edge of the hypo-IRAF, and there were fewer hyper-IRAF spots than hyper-AF spots Figures . The posThe regularity of FFA progression in BCD patients has been reported in detail in our previously published paper . The speμm greater than the first, indicating that EZ was also lost progressively in the right eye of P1. In type 2 patients, the EZ, the interdigitation zone, and part of the RPE were absent in the macular area, and the external limiting membrane was clearly visible . There was no EZ residue in the fovea of the right eye of P1, so the distance between the remnant of the EZ at the temporal side of the fovea and the Bruch membrane at the fovea was measured. The last distance was 165  visible . Except  changes .The 6 patients observed in this study ranged in age from 30 to 46 years, which is the common age of BCD onset. The results of gene mutation of the 6 patients were all BCD-related gene mutation sites , 12.Color fundus photographs of type 1 BCD showed a decrease in crystalline deposits with the progression of disease, consistent with previous reports . The yelIt is known that AF mainly reflects the fluorescence of lipofuscin . The locRetinal and choroidal atrophy in the last FFA examination of 6 BCD patients was significantly worse than that in the first FFA examination, which was consistent with previous reports , 9. BothIn this study, the changes in the retina and choroid in BCD patients were quantified by SD-OCT. In type 1 patients, both the retina and choroid thinned significantly, and the diameters of choroidal great vessels narrowed. Studies have reported that the blood flow density of choroidal capillaries in BCD patients decreases as measured by optical coherence tomography angiography . AccordiThe limitation of this study was that the number of eyes included was small and the observation time was not very long. However, if the rarity of the disease and previous literature are considered, this was the first study to observe the progress of BCD by multimodal imaging, and the number of cases reported was relatively large.In conclusion, in addition to atrophy of the retina and choroid capillary layers, the diameter of the choroid great vessels in type 1 BCD was also significantly reduced. In type 2 BCD patients, retinopathy was significantly aggravated, except for the macula area. Noninvasive examination of AF, IRAF, and SD-OCT have great advantages in assessing BCD progression and changes.


Albizia julibrissin Durazz is one of the most common herbs used for depression and anxiety treatment, but its mechanism of action as an antidepressant or anxiolytic drug have not been fully understood. We previously isolated and identified one lignan glycoside compound from Albizia Julibrissin Durazz, (-)-syringaresinol-4-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside (SAG), that inhibited all three monoamine transporters with a mechanism of action different from that of the conventional antidepressants. In this study, we generated homology models for human dopamine transporter and human norepinephrine transporter, based on the X-ray structure of Drosophila dopamine transporter, and conducted the molecular docking of SAG to all three human monoamine transporters. Our computational results indicated that SAG binds to an allosteric site (S2) that has been demonstrated to be formed by an aromatic pocket positioned in the scaffold domain in the extracellular vestibule connected to the central site (S1) in these monoamine transporters. In addition, we demonstrated that SAG stabilizes a conformation of serotonin transporter with both the extracellular and cytoplasmic pathways closed. Furthermore, we performed mutagenesis of the residues in both the allosteric and orthosteric sites to biochemically validate SAG binding in all three monoamine transporters. Our results are consistent with the molecular docking calculation and support the association of SAG with the allosteric site. We expect that this herbal molecule could become a lead compound for the development of new therapeutic agents with a novel mechanism of action. Albizia julibrissin Durazz is one of the most popular herbs that has been historically used for depression and anxiety treatment in traditional Chinese medicine. The major ingredients in Albizia Julibrissin Durazz include triterpenoids, lignans, flavonoids, saponins, and sterols , dopamine transporter (DAT), and norepinephrine transporter (NET). They terminate monoaminergic signaling by the reuptake of monoamine neurotransmitters from the synaptic cleft in the central nerve system . Among tThe previously resolved high-resolution structures of DAT and SERT bound with their specific inhibitors have provided structural insights into the molecular basis for antidepressant action on the monoamine transporters . In thesAlbizia julibrissin Durazz that acted on SERT by a novel underlying mechanism of action different from that of SSRIs -syringaresinol-4-O-D-apiofuranosyl-(1→2)-D-glucopyranoside (SAG) and (-)-syringaresinol-4,4′-bis-O-D-glucopyranoside (SBG), from the bark of of SSRIs . Our preof SSRIs . Of thesof SSRIs .In the present work, we conducted the molecular docking of SAG to the S2 site in the structure of human SERT (hSERT) and homology models of human DAT (hDAT) and human NET (hNET). In addition, we investigated the effect of SAG on conformation of the cytoplasmic pathway in SERT. Furthermore, we performed mutagenesis of the residues in the allosteric and orthosteric binding sites to biochemically validate SAG binding in all three monoamine transporters. Our results provided new insights into the association of SAG with the S2 site and also the specificity of SAG inhibition of monoamine transporters.+), 4-[4-(dimethylamino)styryl]-N-methylpyridinium (ASP+), fluoxetine, GBR12909, and desipramine were purchased from Sigma-Aldrich. Lipofectamine 2000 and Micro BCA protein assay reagent kit were obtained from Thermo Fisher Scientific. 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA) was purchased from Biotium. All other reagents were of analytical grade.(-)-Syringaresinol-4-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside (SAG) was purchased from Chengdu HerbSubstance Biotechnology Co. Ltd., Sichuan, China (batch no. PCS0316). HeLa cells (CCL-2) were from American Type Culture Collection. The expression plasmids for hSERT, hDAT, and hNET were from the Dr. Rudnick lab . The S277C/X5C expression plasmid used in this study was described previously . 4-[4-(DDrosophila melanogaster DAT (dDAT). The alignment between two sequences was obtained with ClustalW and covered residues 58 to 198 and 209 to 601 for hDAT or 54 to 187 and 199 to 598 for hNET, that is, all except a portion of extracellular loop 2 and the N and C termini. The sequence identity between the target and the template is 52% for hDAT or 55% for hNET, respectively. The models with the highest scores were selected for visualization. Figures of structures and models were generated using PyMOL v2.5.2 .Homology models of hDAT and hNET were generated with Modeller  based onMolecular docking was carried out with Glide software in Schrödinger Suites v2021.2 on the structure of hSERT  and the models for hDAT and hNET. The 15B8 Fab (chains B and C) and ligands such as 5-HT, acetylglucosamine, cholesterol, pentane, heptane, decane, and dodecane present in the hSERT structure were removed. The template structure was then subjected to automated structure preparation using the Protein Preparation Wizard in order to optimize the hydrogen bonding network, conformation of bonds, and energy constraints. Ligand preparation was preformed using Ligprep. The SAG molecule was input into Maestro and optimized for its conformation and energy in the OPLS4 force field . ProtonaThe mutants used for this study were constructed in the WT background carried by the expression plasmids for hSERT, hDAT, or hNET under the control of CMV promotor in pcDNA3.1 vector, respectively. All mutants were generated using the Mut Express II Fast Mutagenesis Kit (Vazyme) and confirmed by full-length DNA sequencing.2 incubator. Cells were plated in a 6-well culture plate and grown overnight. Cells at ∼70% confluence were transfected with hSERT, hDAT, or hNET cDNA in pcDNA3.1 by lipofectamine 2000. Transfected cells were incubated for 24 h at 37°C with 5% CO2 and then transferred into a 12-well plate placed with polylysine coated slides. After grown for additional 12–16 h, the cells were assayed for APP+ or ASP+ fluorescence image acquisition.HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified 5% CO2, 1.2 mM MgSO4, and 0.1% (w/v) glucose. APP+ uptake was measured by adding 500 μl KRH buffer containing 2 μM APP+ and incubating for 5 min at room temperature. Excess APP+ was then removed by rapid washing three times with 500 μl KRH buffer. The extent of APP+ accumulated in the cells was determined by confocal imaging analysis. ASP+ binding to SERT in the cell membrane was measured with digitonin-permeabilized cells .Images were acquired at room temperature using a 20 or ×60 water immersion objective with the Zeiss LSM 900 confocal microscope . APP+ anNonlinear regression fits of experimental and calculated data were performed with Origin (Origin Lab). The statistical analysis given was from multiple experiments. Data with error bars in the figures represent the mean ± SD for at least 10 measurements per condition in one experiment or the mean ± SEM from at least three experiments as indicated, respectively. Statistical analysis was performed using One-Way ANOVA under SPSS 26.0.Drosophila DAT . The sequence identity between the target and dDAT is 52% for hDAT or 55% for hNET, respectively, and therefore we expect that the Cα trace of either hDAT or hNET is accurate to within 1 Å of the native structure (+ and Cl− ions in the binding site were modeled on those observed in the dDAT template structure (+ and green for Cl−). The substrate dopamine or norepinephrine was also modeled into the central binding site (S1) of hDAT or hNET, respectively, based on the dopamine molecule observed in dDAT  formed by an aromatic pocket positioned in the scaffold domain in the extracellular vestibule . The subOur previous study has demonstrated that SAG noncompetitively inhibited SERT activity . To examWe previously investigated the influence of SAG on the outward-facing conformation in SERT by using accessibility measurement of a strategically positioned cysteine residue, Y107C, in the extracellular substrate permeation pathway . Our res+ binding to SERT.We previously demonstrated that the cysteine residue in the cytoplasmic pathway, S277C, reacted with the cysteine reagent MTSEA more when the cytoplasmic pathway is open, and less when the pathway is closed . These m+ binding in the cell plasma membrane of SERT mutant S277C in the presence of 25 μg/ml digitonin  that inactivated ∼50% of ASPanalysis .+ and Cl−  SAG associates with the S2 site of hSERT in the molecular docking; 2) mutating residues in the S2 site but not the S1 site leads to dramatical reductions in SAG inhibition of hSERT transport and ligand binding; 3) SAG stabilizes a conformation of hSERT with both the extracellular and cytoplasmic pathways closed . Taken tAlthough SAG strongly inhibited hSERT activity, the herbal molecule also exerted an inhibitory effect on both hDAT and hNET. Its IC50 values in hDAT and hNET were 11 or 16 folds higher than that in hSERT, indicating that SAG is slightly selective in antagonizing hSERT over hDAT or hNET. The higher selectivity of SAG in inhibiting hSERT was also evidenced by our experimental results of site-directed mutagenesis of the S2 site in these monoamine transporters. Replacement of the S2 residues in hSERT, such as D328A, E494Q, T497A, P560G, P561G, Q562N, Y579A, and T583A, led to profound reductions in SAG inhibition potency by more than 40 folds , whereasTo compare SAG binding in all three monoamine transporters, we superimposed the molecular docking models of the hSERT-SAG, the hDAT-SAG, and the hNET-SAG complexes . hDAT anTaken together, we present here that the herbal molecule, SAG, binds to the allosteric site of monoamine transporters through the various interactions with various residues, which opens for the possibility of developing novel antidepressant drugs with distinct pharmacodynamic profiles and specificities. Although SAG strongly inhibits monoamine transporters and exerts a specificity for hSERT, its inhibition potency and selectivity are lower than those of SSRIs. It is notable that the allosteric site of monoamine transporters has been underexplored in structure-based drug design . We anti


Urogenital malignancy accounts for one of the major causes of cancer-related deaths globally. Numerous studies have investigated novel molecular markers in the blood circulation, tumor tissue, or urine in order to assist in the clinical identification of tumors at early stages, predict the response of therapeutic strategies, and give accurate prognosis assessment. As an endogenous inhibitor of lysosomal cysteine proteinases, cystatin C plays an integral role in diverse processes. A substantial number of studies have indicated that it may be such a potential promising biomarker. Therefore, this review was intended to provide a detailed overview of the role of cystatin C in urogenital malignancy. Urogenital malignancies are a spectrum of fatal cancers that affect the urinary and/or reproductive organs , 2. ProsCystatin C is stably expressed in all nucleated cells and is involved in various physiological or pathological conditions . It is oCystatin is a natural cysteine protease inhibitor that is widely present in the human body . The cysCystatin C, as the most potent known inhibitor of cysteine peptidases , stronglIn addition to the aforementioned regulation of intracellular and extracellular lysosomal cysteine proteins, cystatin C also exerts a variety of important functions , 14, inc2 at follow-up, whereas serum creatinine was not. They found that when postoperative cystatin C levels were elevated in the range of 0.9-1.0 mg/l, postoperative creatinine levels remained stable at around 1.3 mg/dl, while cystatin C and creatinine levels remained highly correlated when postoperative cystatin C levels were below 0.9 mg/l or above 1.0 mg/l. The authors referred to the postoperative plateau period of 1.2-1.3 mg/dl of creatinine as the “creatinine blind area” during which fluctuations in cystatin C levels might better predict renal impairment. Within the “creatinine blind area”, postoperative renal impairment is undetectable in up to one-third of patients, yet within this range, significant changes in serum cystatin C levels can be observed in these patients. The finding may change the traditional definition of creatinine-based acute kidney injury (AKI).Preoperative and postoperative serum cystatin C levels may offer potential predictive value for postoperative renal function impairment in RCC patients , 47, 48.Three studies evaluated the predictive value of serum cystatin C level on RCC patients’ prognosis , 41, 49.In 1995, Jacobsson et al.  investigLane et al.  attempte177Lu] Lu-PSMA-617 radioligand and showed that cystatin C was one of the most reliable predictive markers of nephrotoxicity. At baseline, serum cystatin C was elevated in only 14 patients. However, cystatin C was elevated in 32 patients (58%) after treatment. Yang et al.  in patients with testicular cancer, Ichioka et al.  attempteTan et al.  investigCystatin C is a biomarker widely present in human serum, tissues, and urine. Numerous studies have shown its promising clinical application as a predictor of renal function and tumor prognosis. A large number of previous studies, including in the field of urogenital malignancy Table 1,JW was responsible for the study design. LD and ZL participated in the collection and assembly of literature. LD and JW conducted the literature analysis and interpretation. The manuscript was drafted by LD. JW proofread the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version.