N,N,N′-trisubstituted thioureas, with varying substitution patterns, for reactivity-controlled nanocrystal synthesis are reported.Crystal structures of three  N,N,N′-trisubstituted thioureas are reported, namely N,N,N′-tribenzylthiourea, C22H22N2S (1), N-methyl-N,N′-diphenylthiourea, C14H14N2S (2), and N,N-di-n-butyl-N′-phenylthiourea, C15H24N2S (3). The influence of the different substituents on the thioureas is clear from the delocalization of the thiourea C—N and C=S bonds, while the crystal structures show infinite chains of N,N,N′-tribenzylthiourea (1), hydrogen-bonded pairs of N-methyl-N,N′-diphenylthiourea (2) and hexamer ring assemblies of N,N-di-n-butyl-N′-phenylthiourea (3) molecules. The above-mentioned compounds were synthesized via a mild, general procedure, readily accessible precursors and with a high yield, providing straightforward access to a whole library of thioureas.The synthesis and single-crystal X-ray structures of three  These thioureas are synthesized via a one-step click reaction between isothiocyanates and primary or secondary amines , N-methyl-N,N′-diphenylthiourea (2), and N-phenyl-N′,N′-di-n-butylthiourea (3), prepared via a simple, straightforward synthesis method making use of readily commercially available compounds, to a high purity (> 99%) and with a high yield (> 75%).Herein, we report the single-crystal X-ray structural analysis of the following trisubstituted thioureas: 1 crystallizes in the centrosymmetric monoclinic space group P21/c, with the asymmetric unit consisting of one N,N,N′-tribenzylthiourea molecule. On the one hand, the secondary amine benzyl ring (C3–C8) is found to be almost completely parallel to one of the tertiary amine benzyl rings (C17–C22), subtending a dihedral angle of 8.92 (8)° between the best planes through the two benzene rings. On the other hand, the two tertiary amine benzyl rings (C10–C15 and C17–C22) are highly twisted to each other, with a dihedral angle of 76.96 (7)° between the best planes through the two benzene rings . The N1—C1 and C1—N2 bond distances are 1.3419 (18) and 1.3569 (18) Å, respectively, while the C1=S1 (double) bond distance is 1.6905 (14) Å.Compound gs Fig. 1a. The N2 crystallizes in the centrosymmetric triclinic space group PN-methyl-N,N′-diphenylthiourea molecules in the asymmetric unit. The secondary and tertiary amine phenyl rings  subtend a dihedral angle of 69.39 (9) and 75.70 (10)°, respectively, between the best planes through the two phenyl rings . The N1—C1 and C1—N2 bond distances are 1.359 (2) and 1.352 (3) Å, for molecule 1, while the respective N21—C21 and C21—N22 bond distances are 1.367 (2) and 1.345 (3) Å, for molecule 2. The C1=S1 and C21=S22 (double) bond distances are 1.6835 (17) and 1.6798 (19) Å, for molecule 1 and 2, respectively.Compound gs Fig. 1b. The N1, i.e. the lone electron pair on N1/N21 is more delocalized towards the secondary amine phenyl ring substituent in 2, leading to an increased N1—C1/N21—C22 distance of 1.359 (2)/1.367 (2) Å, which is even more pronounced for the second molecule in the asymmetric unit, because of higher planarity of the phenyl ring with the N—C(=S)—N plane /1.6798 (19) Å in comparison with 1.The influence of the two phenyl substituents on the delocalization of the N1—C1, C1—N2 and C1=S1 bonds is clear, in comparison with the structure of ne Fig. 2. However3 has very recently been deposited with the Cambridge Structural Database (CSD) °, while the two butyl substituents are found completely staggered . The N1—C1 and C1—N2 bond distances are 1.3594 (15) and 1.3432 (15) Å, respectively, while the C1=S2 (double) bond distance is 1.7004 (11) Å. The delocalization of N1 towards the secondary amine phenyl substituent is also noticed here, comparable to 2, while there is minimal delocalization of N2 towards the butyl substituents, consequently showing the shortest C1—N2 and the longest C1=S1 distances.The structure of  al. 2021 might beed Fig. 1c. The N1, only weak π–π interactions are present in the crystal packing, with rather large centroid–centroid distances ranging from 4.4279 (11) to 5.9248 (9) Å. However, clear intermolecular hydrogen bonds are found between the secondary amine N1—H1 hydrogen atoms and the thiourea S1 atoms  direction, and forming columnar arrangements through alternating orientations of the molecules –3.8507 (19) Å, complementing the crystal packing.Despite the presence of three benzyl moieties in the molecular structure of es Fig. 3. Non-cla1, the presence of two phenyl substituents in the molecular structure of 2, only leads to weak π–π interactions present in the crystal packing, with rather large centroid–centroid distances ranging from 4.8431 (13) to 5.9503 (12) Å. However, in this case, intermolecular hydrogen bonds are formed between the two distinct molecules in the asymmetric unit, i.e. between the secondary amine N1—H1 hydrogen atom of the first molecule and the thiourea S21 atom of the second molecule . Furthermore, several C—H⋯π contacts are observed in the range of 3.518 (2)–3.800 (2) Å, complementing the crystal packing.Analogous to rs Fig. 4. Non-cla1 and 2, for 3, only one type of weak π–π interaction is present in the crystal packing, i.e. between symmetry-equivalent phenyl substituents, with a centroid–centroid distance of 4.9098 (10) Å. Intermolecular hydrogen bonds are formed between the secondary amine N1—H1 hydrogen atoms and the thiourea S1 atoms . Only one C—H⋯π contact is observed .Analogous to 1, 2 and 3, deposited with the Cambridge Structural Database -N,N-dimethylthiourea  was purchased from Alfa Aesar. Chloroform-d1  was purchased from Carl Roth. Toluene (99.0%), acetonitrile (99.9%), n-hexane (99.0%), and abs. ethanol (99.8%) were purchased from Chem-Lab. Benzyl isothiocyanate (98.0%), dibenzylamine (97.0%), N-methylaniline (98.0%), and di-n-butylamine (99.5%) were purchased from Sigma-Aldrich. Dichloromethane-d2 (99.8%D) was purchased from VWR. The thioureas were synthesized according to the procedure by Hendricks and Co-workers on a 30 mmol scale with the addition of a recrystallization step to purify the thiourea : A 40 mL vial was loaded with benzyl isothiocyanate  in toluene (5 mL). To this, a solution of dibenzylamine  in toluene (5 mL) was added dropwise. The mixture was left to stir for 1 h at room temperature. Afterwards, the solvent was removed under reduced pressure, and the residual solid was recrystallized from hot acetonitrile which was cooled slowly (> 2 h) to room temperature and then to refrigerator temperature . The formed crystals were filtered off and extensively dried under dynamic vacuum to obtain white needle-like crystals , suitable for single-crystal X-ray diffraction analysis. 1H NMR : δ 7.45–7.15 , δ 7.10–6.95 , δ 5.80 , δ 5.00 , 4.80 . 13C NMR : δ 183.16, 137.78, 136.06, 129.17, 128.75, 128.03, 127.62, 127.19, 54.37, 50.79. LC–MS (API–ES) calculated for C22H23N2S [M+H]+ 347.16, found 347.1.Synthesis ofN-methyl-N,N’-diphenylthiourea (2): A 40 mL vial was loaded with phenyl isothiocyanate  in toluene (5 mL). To this, a solution of N-methylaniline  in toluene (5 mL) was added dropwise. The mixture was left to stir for 6 h at 323 K, since the reaction with aniline derivatives elapses more sluggishly. Afterwards, the solvent was removed under reduced pressure, and the residual solid was recrystallized from a hot hexane:ethanol (10:1) mixture which was cooled slowly (> 2 h) to room temperature and then to refrigerator temperature . The formed crystals were filtered off and extensively dried under dynamic vacuum to obtain white needle-like crystals , suitable for single-crystal X-ray diffraction analysis. 1H NMR : δ 7.55–7.50 , δ 7.45–7.35 , δ 7.32–7.27 , δ 7.20–7.12 , δ 7.00 , δ 3.70 . 13C NMR : δ 181.92, 143.52, 140.04, 131.05, 129.03, 128.76, 127.44, 126.21, 126.12, 43.73. LC–MS (API–ES) calculated for C14H15N2S [M+H]+ 243.10, found 243.1.N,N-di-n-butyl-N′-phenylthiourea (3):Synthesis of  A 40 mL vial was loaded with phenyl isothiocyanate  in toluene (5 mL). To this, a solution of di-n-butylamine  in toluene (5 mL) was added dropwise. The mixture was left to stir for 1 h at room temperature. Afterwards, the solvent was removed under reduced pressure, and the residual solid was recrystallized from a hot hexane:ethanol (10:1) mixture which was cooled slowly (> 2 h) to room temperature and then to refrigerator temperature . The formed crystals were filtered off and extensively dried under dynamic vacuum to obtain white needle-like crystals , suitable for single-crystal X-ray diffraction analysis. 1H NMR : δ 7.40–7.28 , δ 7.23–7.15 , δ 7.00 , δ 3.67 , δ 1.71 , δ 1.38 , δ 0.97 . 13C NMR : δ 181.56, 140.65, 128.85, 126.21, 125.84, 51.85, 29.95, 20.69, 14.06. LC–MS (API–ES) calc for C15H25N2S [M+H]+ 265.17, found 265.2.NMR spectroscopy. Nuclear Magnetic Resonance (NMR) spectra of the synthesized organics were recorded on a Bruker 400 MHz. Chemical shifts (δ) are given in ppm and the residual solvent peak was used as an internal standard . The signal multiplicity is denoted as follows: s (singlet), d (doublet), t (triplet), quad (quadruplet), quin (quintet), six (sextet), m (multiplet). Coupling constants are reported in Hertz (Hz). All resonances were corrected prior to integration by subtracting a background from the measured intensity. 1H, and 13C spectra were acquired using the standard pulse sequences from the Bruker library; zg30, and jmod (Attached Proton Test = APT), respectively.Mass spectroscopy. Mass spectra (MS) were measured with an Agilent ESI single quadrupole detector type VL and an Agilent APCI single quadrupole detector type VL.Ueq of the parent atoms. All other hydrogen atoms were refined in the riding mode with isotropic temperature factors fixed at 1.2 times Ueq of the parent atoms (1.5 times for methyl groups).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989022000147/vm2258sup1.cifCrystal structure: contains datablock(s) 1, 2, 3, New_Global_Publ_Block. DOI: 10.1107/S2056989022000147/vm22581sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989022000147/vm22582sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S2056989022000147/vm22583sup4.hklStructure factors: contains datablock(s) 3. DOI: 2132677, 2132676, 2132675CCDC references:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


X  in the crystal structures of the NiII and CuII based complexes fabricated from the sterically crowding cyclohexyl-containing Schiff base dyes. Photophysical properties of the reported complexes are also discussed.This work shows the importance of counterintuitive ultra long distances, intramolecular preagostic attractive C—H⋯Ni/Cu and C—H⋯O, C—H⋯H—C contacts and a bunch of other extremely strong non-classic mainly London dispersion driven intermolecular interactions involving C—H bonds: C—H⋯ II and CuII discrete mononuclear homoleptic complexes [Ni2] and [Cu2] fabricated from the Schiff base dyes o-HOC6H4—CH=N—cyclo-C6H11 (HLI) and o-HOC10H6—CH=N—cyclo-C6H11 (HLII), containing the sterically crowding cyclohexyl units. The six-membered metallocycles adopt a clearly defined envelope conformation in [Ni(LII)2], while they are much more planar in the structures of [Ni(LI)2] and [Cu2]. It has been demonstrated by in-depth bonding analyses based on the ETS-NOCV and Interacting Quantum Atoms energy-decomposition schemes that application of the bulky substituents, containing several C—H groups, has led to the formation of a set of classical and unintuitive intra- and inter-molecular interactions. All together they are responsible for the high stability of [Ni2] and [Cu2]. More specifically, London dispersion dominated intramolecular C—H⋯O, C—H⋯N and C—H⋯H—C hydrogen bonds are recognized and, importantly, the attractive, chiefly the Coulomb driven, preagostic (not repulsive anagostic) C—H⋯Ni/Cu interactions have been discovered despite their relatively long distances (∼2.8–3.1 Å). All the complexes are further stabilized by the extremely efficient intermolecular C—H⋯π(benzene) and C—H⋯π(chelate) interactions, where both the charge-delocalization and London dispersion constituents appear to be crucial for the crystal packing of the obtained complexes. All the complexes were found to be photoluminescent in CH2Cl2, with [Cu(LII)2] exhibiting the most pronounced emission – the time-dependent density-functional-theory computations revealed that it is mostly caused by metal-to-ligand charge-transfer transitions.This work reports on synthesis and extensive experimental and theoretical investigations on photophysical, structural and thermal properties of the Ni To highlight a pivotal role of this type of interaction it is sufficient to mention that the double-helix structure of DNA is dictated by a bench of non-covalent interactions . The conference aimed ‘to highlight the role of non-covalent interactions in synthesis, catalysis, crystal engineering, molecular recognition, medicinal chemistry, biology, materials science, electrochemical immobilization, etc., including also theoretical aspects.’About one and a half centuries ago in his prominent doctoral dissertation, J. D. van der Waals was the first who recognized non-covalent interactions    agostic bonding, cation/anion⋯π interaction and many others phosphorylated thioureas NHP(S)(OiPr)2  and [Cu2], respectively. The obtained complexes seem to be excellent platforms to generate a bunch of non-covalent interactions owing to the presence of aromatic benzene rings, aliphatic cyclohexane rings and metal-containing chelate rings. Theoretical studies are then applied to shed light on the origin of their photophysical properties. Although the crystal structures of [Ni(LI)2] 2] 2]  Fig. 1 were inv2.LI,II as yellow viscous oil. HLI,II were involved in the reaction with a half molar amount of M(CH3COO)2  in ethanol. As a result, discrete mononuclear homoleptic complexes [Ni2] and [Cu2], respectively, were isolated with high yields.A reaction of an equimolar amount of cyclohexylamine and salicyaldehyde or 2-hydroxy-1-naphthaldehyde in ethanol under reflux yielded the Schiff bases HLI)2] and [Cu(LI)2] were found to be isostructural, as shown by the single-crystal X-ray diffraction data , while the crystal structures of [Ni(LII)2] and [Cu(LII)2] were solved in the monoclinic space group P21/n with a half of the complex molecule in the asymmetric unit for all the complexes. In complexes, the metal cation is coordinated by two molecules of the deprotonated ligand LI,II via imine nitrogen atom and phenoxy oxygen atom affording a tetracoordinate environment with the formation of a perfect square-planar coordination geometry as shown by the τ4 descriptor 2] and [Cu(LI)2], while they are much more planar in the structure of [Cu(LII)2] 2] and [Cu(LI)2] are ∼35°, while the same angles in the structure of [Cu(LII)2] are ∼45°. The same angles between the planes formed by the benzene or naphthalene and chelate rings, and cyclohexyl and chelate rings are ∼7–11 and 45°, respectively (Table 1The angles between planes formed by the benzene or naphthyl and cyclohexyl rings corresponding to the same ligand in the structures of [Ni(y Table 1.LI)2] and [Cu2] the same oxygen also forms the second C—H⋯O bond with one of the hydrogen atoms from one of the secondary carbons linked to the tertiary carbon 2], which formation, together with a coordination geometry of chelate cycles, prevents the formation of the second intramolecular C—H⋯O bond. Notably, all crystal structures are further stabilized by intermolecular non-covalent interactions of the C—H⋯π and C—H⋯π(chelate) nature , as well as from the elemental analysis data  2] and [Cu(LI)2], which is caused by the shortest contact being between three atoms, rather than being a direct two-atom contact  , with the shortest de + di ≃ 2.7 Å, and are recognized as characteristic of C—H⋯π nature 2] and [Cu(LI)2] are also characterized by significantly smaller proportions of the H⋯N and H⋯O contacts (Table 3LII)2], while the proportions of the C⋯C, C⋯N, C⋯O and C⋯Cu contacts are quite distinct . This is explained by the formation of π(chelate)⋯π intermolecular interactions , which are assigned to the abovementioned intermolecular C—H⋯M and C—H⋯π(chelate) interactions 2] and [Cu(LI)2], since the corresponding enrichment ratios EHX are close to or even higher than unity (Table 3LII)2], while remaining contacts are impoverished  or attractive (agostic). We have recently discovered  → σ*(C—H)/σ(C—H) → Ni(dz2)] and London dispersion terms 2], a very efficient intramolecular instantaneous stabilization is gained with ΔEint(Ni⋯H) = −11.36 kcal mol−1. It is mainly owing to the attractive Coulomb contribution ΔECoulomb = −10.00 kcal mol−1 and slightly stabilizing exchange-correlation term ΔEXC = −1.36 kcal mol−1 , the Coulomb term appeared to be positive, which led to the overall repulsive (anagostic) C—H⋯Ni interactions  diatomic interaction energies Δ−1 Fig. 4. It is iEint(C⋯H) = −6.99 kcal mol−1 and ΔEint(O⋯H) = −5.89 kcal mol−1 . Recently, the latter has been of particular attention in terms of reconsidering the real nature of steric crowding in bulky species 2] the second Ni⋯H contact, with quite similar length to the first, was revealed, which, however, destabilizes the overall structure, although quite insignificantly owing to an unfavourable Coulomb term and negligible stemming from the exchange-correlation constituent 2] is concerned, quite similar stabilizing intramolecular interactions C⋯H and O⋯H were obtained  = −14.16 kcal mol−1 despite a very long distance of 3.065 Å 2] results in even more efficient preagostic attraction ΔEint(Cu⋯H) = −14.67 kcal mol−1 owing to a shorter distance of 3.015 Å  = −8.81 kcal mol−1 2] using the ETS-NOCV scheme  as opposed to the literature claims on insignificance of this constituent 2] .Finally, we briefly analyzed the intermolecular interactions in the example dimeric model of  and [Cu2] in CH2Cl2 yields yellow and reddish yellow solutions, respectively. In the UV–Vis absorption spectra of the complexes, three regions can be clearly defined. The first region, ranging from 200 to ∼300 nm, contains a set of high intense bands corresponding to intraligand π → π* and n → π* transitions  transitions  calculations. Since the qualitative picture of the electronic transitions is similar for all the complexes, we briefly discuss the data for [Ni(LI)2]. All the complexes remain a square-planar geometry in CH2Cl2 and, in line with the experimental data, the analogous three absorption regions were obtained for all species  → π*, as indicated by the dominant transition #13 with the oscillator strength f = 0.208 a.u.  and #5 (f = 0.088 a.u.), are additionally described by both the ligand-to-ligand and ligand-to-metal charge transfers 2] is remarkably more emissive 2] and [Cu(LII)2] exhibit a broad intense band centred at ∼435–450 nm, while the spectrum of [Cu(LI)2] exhibits a broad band with two maxima at ∼375 and 430 nm 2] and [Cu2], fabricated from the Schiff base dyes o-HOC6H4—CH=N—cyclo-C6H11 (HLI) and o-HOC10H6—CH=N—cyclo-C6H11 (HLII), respectively, each containing a bulky aliphatic fragment, namely cyclohexyl.In summary, we studied structural and photophysical properties of the Nitrans-square-planar geometry. Remarkably, the six-membered metallocycles adopt a clearly defined envelope conformation in [Ni2] and [Cu(LI)2], while they are much more planar in the structure of [Cu(LII)2]. This was found to be clearly associated with the formation of different intra- and inter-molecular contacts, which were deeply characterized by the charge- and energy-decomposition scheme ETS-NOCV as well as the IQA approach. In particular, London dispersion dominated intramolecular C—H⋯O, C—H⋯N and C—H⋯H—C interactions were identified and, predominantly, the attractive, mostly Coulomb driven, C—H⋯Ni/Cu preagostic (not repulsive anagostic) bonds were discovered despite their long distances (∼2.8–3.1 Å). Interestingly, despite the long distances, non-negligible charge-delocalization constituent was discovered. Notably, all the crystal structures are further stabilized by very efficient  intermolecular C—H⋯π(benzene) and C—H⋯π(chelate) interactions, which are responsible for their high stability as seen from the thermogravimetric (TG) analyses. Although they contain the prevailing dispersion constituent, the charge-delocalization contribution is only slightly less important followed by the Coulomb term. Our results, clearly showing that the bulky cyclohexyl groups are the sources of London dispersion stabilization, are in line with the recent discoveries outlining the true character of steric effects in small and sizable species  transitions, as indicated by the time-dependent density functional theory (TDDFT) computations. Importantly, all the complexes were found to be planar and photoluminescent in CH2Cl2, with [Cu(LII)2] exhibiting the most pronounced emission, mostly owing to MLCT transitions.Finally, dissolving crystals of the complexes in CH4.4.1.All reagents and solvents were commercially available and used without further purification.4.2.3 were obtained on a Bruker AVANCE II 400 MHz spectrometer at 25°C. Chemical shifts are reported with reference to SiMe4. Infrared spectra (KBr) were recorded with a FT-IR FSM 1201 spectrometer in the range 400–3400 cm–1. UV–Vis and fluorescent spectra from the freshly prepared solutions (5 × 10−5 M) in freshly distilled CH2Cl2 were recorded on an Agilent 8453 instrument and a RF-5301 spectrofluorophotometer. TG analyses were performed by a NETZSCH STA 449 F5 Jupiter instrument in a dynamic air or argon atmosphere (100 ml min−1) from laboratory temperature to 1000°C with a 10°C min−1 heating rate. Microanalyses were performed using a ElementarVario EL III analyzer.Nuclear magnetic resonance (NMR) spectra in CDCl4.3.LI, the solvent and non-reacted starting materials were removed in vacuo. The resulting yellow viscous oil was analyzed and used as is. The resulting solution of HLII was allowed to cool to room temperature to give crystals, which were filtered off.A solution of an equimolar amount of salicylaldehyde or 2-hydroxy-1-naphthaldehyde  and cyclohexylamine  in ethanol (50 ml) was stirred for 1 h under reflux. For a solution of Ha) HLI. Yield = 1.809 g (89%). 1H NMR: δ = 1.28–1.75 , 1.80–1.95 , 3.25–3.35 , 6.84 , 6.96 , 7.32 , 7.38 , 8.37  and 13.30 . Analysis calculated for C13H17NO (203.29): C = 76.81, H = 8.43 and N = 6.89%; found: C = 76.68, H = 8.37 and N = 6.94%.(b) HLII. Yield = 2128 g (84%). 1H NMR: δ = 1.30–1.76 , 1.86–1.97 , 2.03–2.12 , 3.44–3.58 , 6.94 , 7.26 , 7.46 , 7.64 , 7.71 , 7.87 , 8.77 , 14.58 . Analysis calculated for C17H19NO (253.35): C = 80.60, H = 7.56 and N = 5.53%; found: C = 80.48, H = 7.62 and N = 5.48%. in ethanol (10 ml) was added a solution of Ni(CH3COO)24H2O  or Cu(CH3COO)2  in a mixture of water (1 ml) and ethanol (50 ml). The mixture was stirred at room temperature for 1 h. The resulting precipitate was filtered off, washed with ethanol (3 × 50 ml) and dried in vacuo. Then the product was dissolved in CH2Cl2. X-ray suitable crystals were formed during the next few days upon slow evaporation of the solvent.To a solution of HLI)2]. Light brown needle-like crystals. Yield = 0.389 g (84%). Analysis calculated for C26H32N2NiO2 (463.25): C = 67.41, H = 6.96 and N = 6.05%; found: C = 67.52, H = 7.05 and N = 5.97%.(i) [Ni(LI)2]. Dark red block-like crystals. Yield = 0.360 g (77%). Analysis calculated for C26H32CuN2O2 (468.10): C = 66.71, H = 6.89 and N = 5.98%; found: C = 66.62, H = 6.79 and N = 5.91%.(ii) [Cu(LII)2]. Green needle-like crystals. Yield = 0.439 g (78%). Analysis calculated for C34H36N2NiO2 (563.37): C = 72.49, H = 6.44 and N = 4.97%; found: C = 72.61, H = 6.49 and N = 5.02%.(iii) [Ni(LII)2]. Greenish yellow needle-like crystals. Yield = 0.472 g (83%). Analysis calculated for C34H36CuN2O2 (568.22): C = 71.87, H = 6.39 and N = 4.93%; found: C = 71.98, H = 6.34 and N = 4.88%.(iv) [Cu(4.5.X-ray powder diffraction for a bulk sample was carried out using a Rigaku Miniflex X-ray powder diffractometer (λ = 1.54059 Å).4.6.Kα radiation (λ = 0.71073 Å). The data were scaled using the frame-scaling procedure in the X-AREA program system 2]. C26H32N2NiO2, Mr = 463.24 g mol−1, T = 173 (2) K, triclinic, space group P-1 (No. 2), a = 6.4256 (6), b = 7.7129 (8), c = 11.9856 (11) Å, α = 98.709 (8), β = 101.800 (8), γ = 104.300 (8)°, V = 550.52 (10) Å3, Z = 1, ρ = 1.397 g cm−3, μ(Mo Kα) = 0.907 mm−1, reflections = 6716 collected and 2530 unique, Rint = 0.0361, R1 = 0.0592, wR2 = 0.1094 and S = 1.151.(1) Crystal data for [Ni(LI)2]. C26H32CuN2O2, Mr = 468.07 g mol−1, T = 173 (2) K, triclinic, space group P-1 (No. 2), a = 6.4641 (4), b = 7.7224 (5), c = 11.9925 (7) Å, α = 97.647 (5), β = 101.861 (5), γ = 105.261 (5)°, V = 553.99 (6) Å3, Z = 1, ρ = 1.403 g cm−3, μ(Mo Kα) = 1.011 mm−1, reflections = 12 420 collected and 3073 unique, Rint = 0.0206, R1 = 0.0252, wR2 = 0.0693 and S = 1.103.(2) Crystal data for [Cu(LII)2]. C34H36N2NiO2, Mr = 563.36 g mol−1, T = 173 (2) K, monoclinic, space group P21/n, a = 6.0847 (3), b = 10.5704 (7), c = 20.8597 (11) Å, β = 97.882 (4)°, V = 1328.97 (13) Å3, Z = 2, ρ = 1.408 g cm−3, μ(Mo Kα) = 0.766 mm−1, reflections = 15 012 collected and 2930 unique, Rint = 0.043, R1 = 0.0527, wR2 = 0.0879 and S = 1.106.(3) Crystal data for [Ni(LII)2]. C34H36CuN2O2, Mr = 568.19 g mol−1, T = 173 (2) K, monoclinic, space group P21/n, a = 11.0325 (10), b = 5.6889 (3), c = 21.554 (2) Å, β = 99.410 (7)°, V = 1334.59 (19) Å3, Z = 2, ρ = 1.414 g cm−3, μ(Mo Kα) = 0.854 mm−1, reflections = 10 452 collected, 2485 unique, Rint = 0.032, R1 = 0.0455, wR2 = 0.0842 and S = 1.137.(4) Crystal data for [Cu ba37, ba36, ba43, ba41. DOI: 10.1107/S2052252521000610/ed5022sup2.pdfSupporting information. DOI: 1959601, 1972277, 1972278, 2056955CCDC references:


Additionally, we examined whether rosmarinic acid (RA), a natural polyphenol antioxidant, can modulate these processes. Our results show that Aβ1–40-induced platelet adhesion is mediated through NADPH oxidase/ROS/PKC-δ/integrin αIIbβ3 signaling, and these signaling pathways are significantly inhibited by RA. Collectively, these results suggest that RA may have beneficial effects on platelet-associated vascular pathology in AD.In platelets, oxidative stress reportedly increases platelet adhesion to vessels, thus promoting the vascular pathology of various neurodegenerative diseases, including Alzheimer’s disease (AD). Recently, it has been shown that β-amyloid (Aβ) can increase oxidative stress in platelets; however, the underlying mechanism remains elusive. In the present study, we aimed to elucidate the signaling pathway of platelet adhesion induced by Aβ Accumulating evidence suggests a correlation between vascular pathology and Alzheimer’s disease (AD) ,2. In nu2O2 and O2−, regulate platelet functions such as platelet aggregation and adhesion [Platelets are key cells that play a critical role in vascular pathology via thrombogenic activity, as well as by adhering to damaged vessels ,7,8. Recadhesion ,12. Theradhesion ,14,15,16adhesion , and Aβ adhesion . Collectadhesion ,19.IIbβ3, a major adhesion molecule in platelets. We also investigated the underlying mechanism of RA in terms of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and protein kinase C (PKC), which are major signaling molecules involved in integrin activation in platelets.Rosmarinic acid (RA) is one of the major compounds commonly found in species of the family Boraginaceae and the subfamily Nepetoideae (Lamiaceae), and it is known to exhibit various biological activities, including antioxidative and anti-inflammatory effects ,21. PrevTM Green 5-chloromethylfluorescein diacetate (CMFDA), 2,7-dichlorodihydro fluorescent diacetate (H2DCFDA), and dihydroethidium (DHE) were purchased from Thermo Fisher Scientific . Fluorescein isothiocyanate (FITC) anti-human CD41a (integrin αIIb) antibody and APC anti-human CD61 (integrin β3) antibodies were purchased from BD Pharmingen . All other chemical reagents were purchased from Sigma-Aldrich and were of analytical or high-performance liquid chromatography (HPLC) grade.RA was purchased from Sigma-Aldrich Co. , and Trolox was purchased from Tocris Bioscience . The following commercial antibodies were used: PKC-α, PKC-βI, PKC-βII, PKC-γ, PKC-δ , and β-actin . VAS2870 was purchased from Enzo Life Sciences . Gö6976, rottlerin, and lucigenin were purchased from Sigma-Aldrich. Eptifibatide was purchased from Millipore . CellTrackerg for 10 min at 22 °C without breaking platelet pellets. The supernatant obtained was termed platelet-poor plasma. The obtained platelet pellet was washed twice with Tyrode’s buffer (pH 7.4).Freshly collected platelet-rich plasma (PRP) samples from healthy volunteers were procured from the Korean Red Cross Center, a blood donation facility for research purposes. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Ajou University (Project No. 202002-HM-EX-001). PRP was centrifuged at 1000× 1–40; Abcam, San Francisco, CA, USA) peptides were dissolved in ammonium hydroxide  and then mixed with phosphate-buffered saline  to obtain a 1 mg/mL solution. Aliquots were immediately stored at −80 °C and centrifuged for 15 min at 17,000× g prior to use to remove pre-aggregated materials.Briefly, Aβ protein fragment 1–40 (Aβ6 cells/mL) were added to a glass-bottom dish coated with fibronectin . After incubation for 1 h, nonadherent platelets were removed by washing with PBS. Platelets were captured in five fields per dish using a confocal microscope . Adherent cells were calculated by expressing the areas of adherent platelets as a percentage of the total area. This experiment was repeated a total of three or five times.To observe platelet adhesion, stimulated platelets were stained with 10 μM CMFDA for 30 min at 37 °C. After washing with PBS, stained platelets (5 × 106 cells/mL) were added to a glass-bottom dish. After incubation for 1 h, the image of live cells was acquired through differential interference contrast (DIC) imaging using a confocal microscope at 400–1000× magnification . Filopodia, membrane protrusions supported by bundles, were randomly selected per each platelet. The filopodia length and spread area (excluding filopodia) were measured from 20 randomly selected fields with ImageJ software. This experiment was repeated a total four times.For the measurement of the filopodial length and spread area of platelets, a glass-bottom dish was coated with fibronectin . After washing with PBS, treated platelets (5 × 108 cells) were activated with 10 µM Aβ1–40 for 1 h at room temperature and then washed twice with Tyrode’s buffer and HEPES buffer. Next, the cells were immediately fixed with 2% paraformaldehyde on ice for 10 min. The cells were then washed twice in 1 × PBS, followed by the addition of 5 µL of FITC anti-human CD41a (integrin αIIb) or 5 µL of APC anti-human CD61 (integrin β3) to each sample and incubation for 30 min at room temperature in the dark. Finally, the cells were washed with 1 × PBS, resuspended in FACS sheath fluid, and analyzed using an FACSAria III flow cytometer . This experiment was repeated a total of seven times.In brief, the washed human platelets  assay as previously described. The protocol to assess DPPH radical scavenging activity was adapted from Brand-Williams et al. (1995), with minor changes . Briefly2S2O8 solution (0.0378 g/mL) was added to the ABTS solution, and the mixture was left at room temperature for 12 h in the dark. To perform measurements, the ABTS solution was diluted with ethanol to an absorbance of 0.700 ± 0.05 at 734 nm. Subsequently, 270 μL of the free-radical solution was combined with 20 μL of RA. The absorbance was measured at 734 nm after incubation at room temperature for 30 min in the dark. Antioxidant activity, expressed as a percentage of inhibition, was calculated using a previously established equation. Additionally, the half-maximal inhibitory concentration (IC50) was determined. This experiment was repeated a total of three times.The preformed radical monocation of 2,2’-azinobis was generated as previously described . The ABT7 cells), pretreated with RA, Trolox, or VAS 2870 for 30 min were incubated with either Aβ1–40 for 15 min, followed by incubation with 10 µM H2DCFDA or 10 µM DHE for 30 min. Fluorescence was immediately measured using an FACSAria III flow cytometer  with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. Gated cells  were analyzed for each sample. This experiment was repeated a total of six times.Briefly, washed human platelets (1 × 10The activity of NADPH oxidase was determined in membrane fractions (50 μg of protein) incubated with 1 mM EGTA and 5 μM lucigenin in phosphate buffer (pH 7.0). The assay was initiated by adding 50 μM NADPH to the incubation mixture. Samples were immediately counted using a tabletop luminometer , with sampling performed every 6 s over a 5 min period; the fluorescence values were recorded for over 2 min of stable readings and averaged for each sample. This experiment was repeated a total of seven times.3VO4, 0.5 mM phenylmethylsulphonyl fluoride, 0.01 mM leupeptin, and 0.01 mg/mL aprotinin) for 30 min on ice and then centrifuged at 200,000× g in a Beckman Optima TL Ultracentrifuge  at 4 °C for 30 min. The supernatants  were removed, and the remaining pellets were resuspended in lysis buffer B (lysis buffer A containing 1% Triton X-100) and incubated on ice for 1 h. The suspension was then centrifuged as described above, and the supernatant  was obtained. This experiment was repeated a total of three times.Platelets were lysed using different lysis buffers, and lysates were extracted using different procedures. Briefly, the cells were incubated in lysis buffer A , anti-PKC-βI , anti-PKC-βII , anti-PKC-γ , anti-PKC-δ , or anti-actin  overnight. All samples were analyzed using a LAS 4000 mini . This experiment was repeated a total of three times.Western blotting was performed by modifying a previously described procedure . Brieflyt-tests were performed to examine differences in continuous variables, overall and at each time point, investigated in different comparison groups. Differences were considered statistically significant at p < 0.05.All data are expressed as the mean ± standard error of mean (SEM). Two-tailed 1–40 increased platelet adhesion to fibronectin (418.65% ± 47.39%), and RA suppressed this increase in a concentration-dependent manner (236.57% ± 50.83% (10 μM) and 120.34% ± 12.62% (30 μM)) . Furthermore, Aβ1–40 promoted platelet spreading and filopodia to alter the platelet shape ; these changes were inhibited by RA  and eptifibatide .In the present study, we observed that Aβ(30 μM)) a. To detet shape b. Aβ1–40preading c and filpreading d , and RA (30 μM) suppressed this increase (124.11% ± 12.86%) (1–40 increased the activity of integrin β3 (163.86% ± 7.08%), which was suppressed by RA (132.37% ± 9.66% at 10 μM and 104.05% ± 5.78% at 30 μM)  a. Additit 30 μM) b. These 1–40 increased ROS (H2O2 or O2−) production , and this increase was inhibited by 30 μM RA  and 100 μM Trolox .We examined the in vitro antioxidant activity of RA using the DPPH and ABTS assays. As shown in 1–40-induced platelet adhesion was associated with oxidative stress. We observed that 100 μM Trolox almost completely inhibited the Aβ1–40-induced increase in platelet adhesion (119.98% ± 30.71%)  was suppressed following treatment with Trolox   a. Additictively) b,c.1–40-induced ROS generation. Our results revealed that Aβ1–40 increased NADPH oxidase activity (173.48% ± 11.52%) in platelets; this effect was suppressed by treatment with RA , 10 μM VAS2870 (NADPH oxidase inhibitor), and 100 μM Trolox (114.16% ± 6.83% (30 μM RA), 108.76% ± 16.09% (VAS2870), and 108.59% ± 13.01% (100 μM Trolox))  was significantly inhibited by NADPH oxidase inhibition . Additionally, the NADPH oxidase inhibitor completely suppressed Aβ1–40-induced platelet adhesion (128.23% ± 15.46%) ) a. Additi 15.46%) f. These 1–40. Among the various PKC families, Aβ1–40 only increased the expression of PKC-δ (194.11% ± 27.24%); the increased PKC-δ activity was inhibited by 30 μM RA, 10 μM VAS2870, and 100 μM Trolox pretreatment  (1–40-mediated increases in integrin αIIb and integrin β3 levels (176.73% ± 17.60% and 194.41% ± 26.33%) were significantly decreased by the PKC-δ inhibitor, 1 μM rottlerin , but not by the classical PKC inhibitor, 1 μM Gö6976  (1–40-induced increase in platelet adhesion (191.94% ± 15.42%) was blocked only by rottlerin (108.55% ± 12.25%)  a,b. Addictively) c,d. Simi 12.25%) e. These IIbβ3 signaling in the mechanism of Aβ1–40-induced platelet adhesion. We further revealed that Aβ1–40-induced platelet adhesion is ameliorated by RA through inhibition of these signaling pathways.In the present study, we for the first time demonstrated the involvement of NADPH oxidase/ROS/PKC-δ/integrin α1–40 is the primary blood form of Aβ, contributing to vascular amyloid deposition in AD; Aβ1–42 is the predominant form in neural plaques [1–40 accounts for more than 90% of Aβ produced from APP in the body and is the predominant type released from activated human platelets. Although Aβ1–42 has been found to mediate platelet aggregation, the efficacy of Aβ1–42 on vascular events is suggested to be far less than that mediated by Aβ1–40 [1–40 are relatively scarce when compared with those on Aβ1–42. Previously, we reported that the altered miRNA profile in platelets from patients with Alzheimer’s pathology was similar to that of Aβ1–40-exposed platelets in vitro [1–40-stimulated platelets could play an important role during the process of Alzheimer’s pathology. Consistent with our previous report, the present study revealed that Aβ1–40 increased integrin αIIbβ3 levels in human platelets, and that platelet adhesion to fibronectin was almost completely suppressed by a specific integrin αIIbβ3 inhibitor. Additionally, RA suppressed platelet adhesion and integrin αIIbβ3 activity in a concentration-dependent manner , the level of PKC-δ was remarkably increased by Aβ1–40. Additionally, Aβ1–40-induced PKC-δ activation was suppressed by RA, Trolox, or VAS2870 (1–40-induced increase in integrin αIIbβ3 activity and platelet adhesion; however, Gö6976, a nonspecific inhibitor of PKC-α, -β, and-γ, did not demonstrate this effect. These results suggest that Aβ1–40-induced integrin αIIbβ3 activity could be regulated by PKC-δ, which produces a representative G-protein-coupled receptor signaling cascade. PKC is divided into isotypes depending on whether calcium ions mediate their regulation. PKC-α, -β, and -γ are regulated by calcium ions, whereas PKC-δ acts independently of calcium ions. Thus, it is suggested that Aβ1–40-induced integrin αIIbβ3 activity may be regulated independently of calcium ions.Various cascades regulate integrin αn factor . However VAS2870 . Further1–40-induced platelet adhesion may involve inhibition of NADPH oxidase/ROS/PKC-δ/integrin αIIbβ3 signaling. Although reports have previously indicated a relationship between NADPH oxidase and integrin αIIbβ3 activity in platelets [1–40-induced integrin αIIbβ3 in the process of platelet adhesion. Our study further showed that RA inhibits all these signaling pathways, consequently inhibiting platelet adhesion. These results indicate that integrin αIIbβ3 and NADPH oxidase can be potential therapeutic targets for platelet-associated vascular pathology in AD. Unsurprisingly, however, the most serious side-effects commonly observed with integrin αIIbβ3 antagonists include bleeding and thrombocytopenia. Therefore, more specific integrin inhibitors involved in the pathological mechanism are needed. In addition, NADPH oxidase may exhibit different roles in other cells, thus highlighting the need for platelet-specific NADPH oxidase inhibitors. As for RA, it has been reported to attenuate the pathological function of integrin αIIbβ3, and possess antithrombotic effect against Aβ [Overall, the underlying mechanisms for the inhibitory effect of RA on Aβlatelets ,41, thisainst Aβ ,43,44. T


Innate lymphoid cells (ILCs) have a protective immune function at mucosal tissues but can also contribute to immunopathology. Previous work has shown that the serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1) is involved in generating protective ILC3 cytokine responses during bacterial infection. However, whether mTORC1 also regulates IFN-γ-mediated immunopathology has not been investigated. In addition, the role of mTORC2 in ILC3s is unknown. Using mice specifically defective for either mTORC1 or mTORC2 in ILC3s, we show that both mTOR complexes regulate the maintenance of ILC3s at steady state and pathological immune response during colitis. mTORC1 and to a lesser extend mTORC2 promote the proliferation of ILC3s in the small intestine. Upon activation, intestinal ILC3s produce less IFN-γ in the absence of mTOR signaling. During colitis, loss of both mTOR complexes in colonic ILC3s results in the reduced production of inflammatory mediators, recruitment of neutrophils and immunopathology. Similarly, treatment with rapamycin after colitis induction ameliorates the disease. Collectively, our data show a critical role for both mTOR complexes in controlling ILC3 cell numbers and ILC3-driven inflammation in the intestine. Group 1 ILCs (ILC1s) depend on the transcription factor T-box expressed in T cells (T-bet) and secrete T helper (Th)1 cytokines like interferon (IFN)-γ. ILC2s rely on GATA3 and secrete Th2 cytokines. ILC3s express and depend on the transcription factor RAR-related orphan receptor gamma t (RORγt)3 and can be subdivided into natural cytotoxicity receptor (NCR)+ and NCR− cells1. Similar to Th22 and Th17 cells ILC3s secrete interleukin (IL)−22 and IL-17 upon activation6.Innate lymphoid cells (ILCs) are tissue-resident cells with protective but also inflammatory immune functions. ILCs can be grouped into three subsetsCitrobacter rodentium and contribute to the maintenance of gut tissue homeostasis12. Conversely, an inappropriate activation of ILC3s plays a role in intestinal immunopathology. In particular, the enhanced secretion of IFN-γ, CSF2, and IL-17 by ILC3s is detrimental in various colitis mouse models17. In recombination-activating gene 2 (Rag2)-deficient mice treated with α-CD40 antibodies (Abs) to elicit colitis, the release of IL-23 by myeloid cells activates colonic ILC3s to switch to pro-inflammatory IFN-γ production, thereby promoting the disease18.Both cytokines are found to be protective against intestinal pathogens like 20, as they encompass different scaffolding proteins. RAPTOR (regulatory-associated protein of mTOR) is found in mTORC1 whereas RICTOR (rapamycin-insensitive companion of mTOR) is part of mTORC220. Both molecules are essential for the formation and function of the respective complex. mTORC1 promotes protective immune responses of ILC3s against C. rodentium infection21.mTOR exists in two different complexes, mTORC1 and mTORC2Rag2−/− background we show that IFN-γ release by ILCs and recruitment of IFN-γ-producing inflammatory neutrophils is impaired during colitis. Similarly, treatment of mice with rapamycin after colitis induction ameliorates immunopathology. These results reveal a novel role for mTOR in regulating IFN-γ-driven immunopathology by ILC3s.The extent to which mTOR signaling determines the activation-induced switch of ILC3 to IFN-γ production and immunopathology is unknown. Moreover, the role of mTORC2 for ILC3 cytokine release remains unprobed. In this study, we demonstrate that mTORC1 and mTORC2 differentially regulate ILC3s under steady state and activation conditions. Using mice with mTOR deficiency in ILC3s on Rptor or Rictor in ILC3s were generated and backcrossed to Rag2−/− mice . The deletion of Rptor resulted in a reduction of NCR+ and NCR− ILC3s in the small intestine (SI) at steady state 22. Of note, the reduced number of ILC3s was not a result of defective ILC progenitor development, as common lymphoid progenitor cell (CLP), common helper ILC progenitor (CHILP), and ILC2 progenitor (ILC2p) were normal in the bone marrow (BM) of both KO mice  were mixed with Rag2−/− BM cells (Ly5.1) in a 1:1 ratio and adoptively transferred in Rag2−/− recipients (Ly5.1/Ly5.2) and analyzed 5 weeks later  4 marked by phosphorylated tyrosine at position tyrosin (Y) 693 controls IFN-γ expression following IL-23 stimulationice Fig. . The IL-ice Fig. . We therRag2-deficient mice it was shown that ILC3s contribute to the pathology by IL-23-driven release of IFN-γ16. As the absence of mTOR-mediated signaling reduced ILC3s’ capacity to secrete IFN-γ upon IL-23 stimulation, we next wished to probe whether RptorΔRORγt and/or RictorΔRORγt mice develop colitis following α-CD40 Ab treatment. In litter controls, one single injection with α-CD40 Ab resulted in weight loss, detection of epithelial-derived lipocalin-2 (LPC2) in the stool as a biomarker of tissue damage (at days 4 and 10) and histopathological signs of colitis at day 10 after Ab injection . In RptorΔRORγt and RictorΔRORγt mice weight loss, LPC2 expression and histopathological alterations in the cecum and colon mucosa were less severe than in controls  at day 3 upon colitis induction  during α-CD40-induced colitisRptor or Rictor impaired similar biological functions of ILC3s, with a stronger effect of the deletion of Rptor on ILC3s’ cellularity and proliferation at steady state. The IFN-γ response of ILCs during colitis, however, was similarly reduced in both knockout strains. It is likely that both mTOR complexes cooperate in the regulation of the underlying signaling pathways that promote proliferation or IFN-γ responses by ILC3s. Positive and negative crosstalk between both mTOR complexes has been described41. Whether such positive or negative signals are relevant in the pathways regulating ILC3s needs to be analyzed in further experiments.Interestingly, we noticed throughout our study that the deletion of RictorΔRORγt mice as compared to RptorΔRORγt mice. This difference might be explained by the finding that IL-22, which is normal in RictorΔRORγt but reduced in RptorΔRORγt mice , is involved in wasting disease during α-CD40-induced colitis27.We observed a less pronounced weight loss in ce Figs. , is invoRag2−/− mice, pointing to a potential benefit for inflammatory bowel disease (IBD) patients of therapy with mTORC1 inhibitors. In support of this assumption, the treatment with rapamycin was beneficial for adult and pediatric IBD patients in two case studies43.The results from our colitis model prompted us to test an mTORC1 inhibitor in this model. We found that rapamycin treatment after colitis induction ameliorated the disease in Collectively, our data shed light on the role of mTOR signaling regulating ILC3s numbers at steady state and inflammatory cytokine release during ILC3-driven intestinal immunopathology.), α-CD11c (N418), α-CD19 (6D5), α-CD45R , α-CD90.2 , α-Gr-1 , α-TCR-β (H57-597), α-TCR-γδ (UC7-13D5), α-TER-119 (TER-119), α-NKp46 (29A1.4), α-NK1.1 (PK136), α-KLRG1 (2F1), α-p-STAT3 (Tyr705) (13A3-1), α-RORγt (AFKJS-9 or B2D), α-IFN-γ (XMG1.2), α-TNF (MP6-XT22), α-IL-22 (1H8PWSR), α-p-S6 (Ser235/236) (D57.2.2E), α-p-mTOR (Ser2448) (MRRBY), α-Ly-6G (1A8), α-Ly-6C (HK1.4), α-Ki-67 (B56), α-Eomesodermin (Dan11mag), α-CD45.1 (A20), α-CD45.2 (104), α-CD127 (IL-7Rα) (A7R34), α-CD117 (cKit) (2B8), α-active Caspase-3 (C92-605), α-CD25 (PC61), α-CD45 (30-F11), α-CD135 (FLT3) (A2F10), α-α4β7 (LPAM-1) (DATK32), α-T-bet (eBio4B10), α-Sca-1 (D7), α-STAT3 (Tyr705) (13a3-1), α-STAT4 (Tyr 693) (38/p-Stat4), and α-p-AKT(Ser473) (M89-61) were ordered either form BD Biosciences, Biolegend, eBioscience or Cell Signaling Technology. α-CD40 (FGK45.5) Ab was produced in B-cell hybridoma cells (kindly provided by Antonius Rolink) according to standard methods.α-CD3ε (145-2C11), α-CD4 (RM4-5 or GK1.5), α-CD8α (53–6.7), α-CD11b . The animal experiments received the approval of the Cantonal Veterinary Office of the city of Basel, Switzerland. Organ donors for in vitro experiments were 7–17 weeks of age. For in vivo experiments, age- and sex-matched animals were used. Animals were 8–11 weeks of age at the initiation of in vivo experiments.C57BL/6 WT and Rag2−/− mice44 were purchased from Janvier Labs and Taconic, respectively. RORc(γt)-Cretg mice45 and Rag2−/−Ly5.1 were ordered from The Jackson Laboratory. Rptorfl/fl and Rictorfl/fl mice46 were kindly provided by M.N.H. .RptorΔRORγt mice, RORc(γt)-Cretg mice were crossed with Rptorfl/fl mice and then bred to Rag2−/− mice. To receive RictorΔRORγt mice, RORc(γt)-Cretg mice were bred to Rictorfl/fl mice and backcrossed to Rag2−/− mice. In experiments with RptorΔRORγt mice and RictorΔRORγt mice, we used Cre− littermates as controls. To generate Rag2−/−Ly5.1/Ly5.2 mice, Rag2−/− mice were mated with Rag2−/−Ly5.1 mice. Rorγtfm+ mice were received by breeding RORc(γt)-Cretg mice to Rosa26ReYFP/+ mice47. Rorγtfm+ mice were backcrossed to Rag2−/− mice.To generate For isolation of LP cells of the SI, the SI was dissected and mesenteric tissue was removed. The SI was opened longitudinally, incubated in 30 mM EDTA in calcium-/magnesium-free 1x PBS for 30 min and washed repeatedly in cold 1x PBS to remove epithelial cells, feces and mucus. The tissue was digested four times for 15 min at 37 °C in DMEM (Gibco) containing 0.025 mg/ml DNase I (Roche) and 1 mg/ml Collagenase D (Roche). Before and after each digestion step, the tissue was homogenized and washed with DMEM. Homogenate supernatant was collected through a 100 μM cell strainer and stored in DMEM supplemented with 5% fetal bovine serum (FBS) and 2 mM EDTA. Cell suspension was pelleted, resuspended in 40% of isotonic Percoll , underlaid with 80% isotonic Percoll and separated by gradient centrifugation . The cell interphase was collected, washed and used for direct staining or for sorting.To isolate cLP cells, the colon was opened longitudinally, washed in 1x PBS, cut into pieces and washed four times on a shaker in 5 μM EDTA and 10 mM HEPES in 1x PBS for 20 min at 37 °C. The tissue was transferred into a gentleMACS™ C Tube (Miltenyi Biotec) filled with DMEM supplemented with 0.025 mg/ml DNase I, 1 mg/ml Collagenase D and 0.25 mg/ml Collagenase VIII (Roche) and was incubated 30 min at 37 °C on a shaker. The colon pieces were dissociated using gentleMACS™ Octo Dissociator . Incubation and shredding procedure were repeated once more. The colon homogenate was filtered through a 100 μM cell strainer and subjected to gradient centrifugation as described for SI LP cell isolation.To obtain BM cells, femur and tibia from one hind leg were dissected. Bones were cleaned and ground in a mortar in 3% FBS in 1x PBS. Cells were pelleted, incubated in erythrocyte lysis buffer for 2 min at room temperature and washed in 3% FBS in PBS.48, purified cell supernatant) was used to block unspecific antibody-binding. Cells were stained with fluorochrome-conjugated or biotinylated Abs in 3% FCS in 1x PBS for 30 min at 4 °C. To detect biotinylated Abs, cells were incubated with fluorochrome-conjugated streptavidin for 20 min at 4 °C. Unless otherwise indicated the following lineage (lin) cocktails of antibodies were used for flow cytometry: CD3, CD8, CD11c, CD19, B220, Gr-1, TCRβ, TCRγδ, Ter-119, and for sorting of ILC3: CD3, CD8, CD11c, CD19, B220, Gr-1, TCRβ, TCRγδ, Ter-119, and NK1.1. Dead cells were stained with LIVE/DEAD® Fixable Aqua Dead Cell Stain kit (Molecular Probes) according to the manufacturer’s protocol.FcγRII/III monoclonal Ab . Cytokines were stained with antibodies diluted in permeabilization solution at 4 °C.For the detection of intracellular cytokines, cells were incubated with 10 μg/ml Brefeldin A in complete IMDM medium supplemented with 5% FBS at 37 °C for 2–4 h prior to surface staining and staining with LIVE/DEADTranscription factor staining was performed using Foxp3 Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s protocol.® Fixable Aqua Dead Cell Stain kit supplemented with 20 ng/ml IL-23/IL-1β. After washing, cells were immediately fixed with 2% PFA in PBS for 10 min at 37 °C. After two wash steps, cells were resuspended in BD Perm Buffer III (pre-chilled to −20 °C) and permeabilized 30 min on ice. Cells were washed twice and stained with Abs for 30 min on ice. After two final wash steps, cells were analyzed immediately.For phosphoFlow analysis, sort-purified cells were cultured in complete IMDM medium supplemented with 5% FBS and 20 ng/ml IL-23/IL-1β. After 17 h, cells were washed in PBS and stained with LIVE/DEADCell acquisition was performed with FACS Fortessa (BD Biosciences) and data analysis with FlowJo 10 software (Tree Star).Cells were sorted with FACS Aria (BD Biosciences). Purity of isolated cells was ≥98%.−CD90.2+KLRG1− ILC3s, SI LP, or cLP cells were cultured in complete IMDM medium supplemented with 5% FBS at 37 °C under 95% humidity and with 5% CO2. Cells were treated with 20 ng/ml IL-23 and 20 ng/ml IL-1β where indicated. mTORC1 inhibitor rapamycin and mTOR inhibitor PP242 was added to a final concentration of 10 nM and 1 μM, respectively.Sorted LinRag2−/− recipient mice heterozygous for the congenic marker Ly5.1 and Ly5.2 (Rag2−/−Ly5.1/Ly5.2 mice) were lethally irradiated with two doses of 450 rad from a γ-source (Cs 137 source) with a resting period of 4 h.Rag2−/−Ly5.1 and knockout or control mice (Ly5.2) were sacrificed. Bones from both hind legs were removed, cleaned, and grounded in a mortar containing DMEM. BM cells were filtered through a 70 μm cell strainer, centrifuged  and subjected to erythrocyte lysis as described above. Cells were washed twice in PBS and filtered through a 40 μm strainer. The donor cells from Rag2−/−Ly5.1 mice and knockout or control mice were mixed in a 1:1 ratio. One day after irradiation recipient mice were injected with 1.5 × 107 donor cells/200 μl into the tail vein.Donor Recipient mice received Baytril (570 mg/l drinking water = 85 mg/kg bodyweight) during the first 10 days. Mice were sacrificed 5–10 weeks after experiment initiation.31. Complexes were prepared by mixing 2.5 μg recombinant murine IL-2 (0.67 mg/ml) and 7.5 μg anti-IL-2 mAb JES6-1A12  per mouse. The IL-2/α-IL-2 mixture was incubated at 37 °C for 30 min, adjusted to 200 μl/mouse with sterile PBS and injected into mice. Mice were sacrificed 1 day post injection.IL-2/α-IL-2 complexes were previously described to induce proliferation of T cells and NK cells in vivo in mice after intraperitoneally (i.p.) injection18. Weight and health condition of mice was monitored daily.α-CD40 Ab (Clone FGK45.5) was diluted to 1 mg/ml in sterile PBS. Mice were weighed and i.p. injected with 140 μg α-CD40 AbFor in vivo use, lyophilized rapamycin (LC Laboratories) was resuspended to 250 μg/ml in sterile PBS supplemented with 0.125% dimethyl sulfoxide (DMSO) and 2.5% ethanol. Four hour after α-CD40 Ab treatment, mice were i.p. injected with mock solution (PBS with 0.125% DMSO and 2.5% ethanol) or 2.5 mg/kg rapamycin. Injections were repeated every other day until mice were sacrificed.LP inflammatory infiltration , (3) proportion of affected area , and (4) severity markers . A total histology colitis score, ranging from 0 to 12 was obtained by summing the four parameter scores.Whole cecum and 25% of the proximal colon were extracted, cleaned with PBS and fixed in 4% paraformaldehyde  for 48–72 h. Sample dehydration, paraffine-embedding, and hematoxylin and eosin staining were performed according to standard protocols. Histology colitis scores (0–12) were determined in a blinded fashion by a pathologist. Biopsies were scored by assessing (1) epithelial hyperplasia and goblet cell depletion , (2) Cell culture supernatants were collected at the time points indicated and stored at −80 °C. To determine cytokines in cell culture supernatants, LEGENDplex Multi-Analyte Flow Assay Kit Mouse Th Cytokine Panel (13-plex) (Biolegend) was used according to the manufacturer’s protocol. In short, samples or cytokine standards were mixed with assay buffer, capture antibodies, and detection antibodies and incubated on a plate rocker for 2 h at room temperature in the dark. Streptavidin-PE was added and the plate was incubated 30 min at room temperature in the dark. Beads were centrifuged, washed once with wash buffer, and resuspended in wash buffer. Samples were acquired with Cytoflex S and CytExpert Software and analyzed with LEGENDplex V8.0 Software (VigeneTech). Cytokine concentration was normalized to cell numbers.Fecal samples were diluted in sterile PBS to a final concentration of 0.1 g of feces per 1 ml, incubated for 15 min at room temperature, and disrupted manually. Solid fibers were removed by centrifugation . Feces supernatant were stored at −20 °C.Mouse LPC2/NGAL DuoSet ELISA Kit (R&D Systems) was used according to the manufacturer’s protocol. Optical density at 450 nm was measured using SpectraMax 340 microplate reader (Molecular Devices). Wavelength correction was performed by subtraction of optical density measured at 570 nm.t test or one-way ANOVA with multiple comparison (Bonferroni) were used. Data are shown as mean values ± SD.Prism 8 Software  was used to analyze the normality of data sets and statistical significance. Non-normal distributed data were analyzed with Mann–Whitney U test. For normal distributed data sets two-tailed unpaired Supplementary Information


Following the publication of this article , concern    ○ Fig 3A left ACC-2 ASO-NC and S7B Fig right ACC-2 shR-PRKCH+ASO-miR-24-3p (4) panel    ○ Fig 3A right ACC-M pcDNA3 and S7B Fig left ACC-M shR-PRKCH+ASO-miR-24-3p (4) panel    ○ Fig 3A right ACCM ASO-NC and S7B Fig left ACC-M shR-PRKCH+ASO-NC (3) panel    ○ Fig 3B ACC-2 ASO-NC 0hr and Fig 3B ACC-2 ASO-miR-24-3P 0hr    ○ Fig 3B ACC-2 pri-miR-24-3p 48hr and Fig 3B ACC-M pri-miR-24-3p 24hr    ○ The top left GAPDH panel of Fig 5C and the bottom right GAPDH panel of Fig 5C    ○ The right colony assay panel for pSilencer-NC+ASO-NC ofS7 Fig and the right colony assay panel for shR-PRKCH+ASO-miR-24-3p of S7 Fig.The following panels appear similar, but represent different experimental results:    ○ Fig 3A left ACC-M pcDNA3, S7B Fig right ACC-M pSilencer-NC+ASO-NC (1) panel, and S7B Fig right ACC-M shR-PRKCH+ASO-miR-24-3p (4) panel    ○ The left ACC-2 pcDNA3+pcDNA3/PRKCH (2) panel of Fig 6D and the left ACC-M pcDNA3+pcDNA3/PRKCH (2) panel of Fig 6D.    ○ The right ACC-2 pcDNA3+pcDNA3 (1) panel of Fig 6D and the right ACC-2 pri-miR-24-3p+pcDNA3/PRKCH (4) panel of Fig 6D.The following panels partially overlap, but represent different experimental results:The corresponding author explained that duplicate and partially overlapping panels were included inadvertently during figure preparation. In addition to the concerns raised by the journal, the corresponding author clarified that the Fig 5C ACC-2-GAPDH-pSilencer-NC panel, and the S7B Fig ACC-2-pSilencer-NC+ASO-NC, ACC-2-shR-PRKCH+ASO-NC, and ACC-M-pSilencer-NC+ASO-miR-24-3p panels are incorrect and were inadvertently included during the figure preparation.PLOS ONE Editors remain concerned about the reliability of data reporting overall in the article given the number of errors identified. Therefore, the PLOS ONE Editors retract this article.The above concerns impact multiple figure panels representing results obtained from four different types of experiments reported in the article. Underlying data and replacement panels for each figure affected by the image concerns were provided to the journal. While the replacement panels satisfactorily addressed some of the above concerns, not all issues were resolved, and the MZ, JZ, HZ, and HT agreed with the retraction. MZ, JZ, and HZ apologized for the issues with the published article.


Oxya chinensis sinuosa (rice field grasshopper) is an edible insect with numerous health beneficial properties, traditionally being used to treat many ailments in Korea and other countries. O. chinensis sinuosa has been used from centuries, however, a little is known about the chemical functionality of its bioactive compounds. Therefore, this study examined the anti-inflammatory and cathepsin C inhibitory activities of N-acetyldopamine dimer -2--3-acetylamino-7-(N-acetyl-2″-aminoethyl)-1,4-benzodioxane (DAB1) isolated from O. chinensis sinuosa. Results showed that DAB1 reduced the expression of pro-inflammatory mediator  and cytokines , and curtailed the nuclear translocation of NF-κB by inhibiting the phosphorylation of IκBα in lipopolysaccharide stimulated macrophages. Additionally, DAB1 inhibited cathepsin C activity at the cellular level, supported by in vitro assay . Moreover, combinatorial molecular simulation and binding free energy analysis suggested a significant stability and binding affinity of cathepsin C-DAB1 complex via formation of hydrogen bond and hydrophobic interactions with the catalytic residues . Also, essential dynamics analysis showed DAB1 induced non-functional motions in cathepsin C structure. Collectively, DAB1 was concluded as anti-inflammatory and cathepsin C inhibiting agent and could be used in the drug development against respective diseases. In particular, plant-derived products have been studied, concerning various biological activities including anti-inflammatory activity O. chinensis sinuosa using Raw 264.7 murine macrophage cells stimulated by lipopolysaccharide (LPS). Initially, bioactive compounds were isolated from the selected insect (O. chinensis), followed by their anti-inflammatory and in vitro cathepsin C inhibitory profiling. Furthermore, in silico approaches were used to provide insights into intermolecular interaction of the isolated bioactive compounds with cathepsin C.Therefore, this study examined the anti-inflammatory and cathepsin C inhibitory potential of compounds isolated from 22.1All the chemical and reagents of analytical grade were used in the study. Antibody for inducible nitric oxide synthases (iNOS) was obtained from Abcam . Also, antibodies for cyclooxygenase-2 (COX-2), nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha (IκBα), nuclear factor kappa B (NF-κB), interleukin 6 (IL-6), and β-actin were bought from Santa Cruz Biotechnology . The active form of mouse cathepsin C was procured from R&D Systems .2.2O. chinensis sinuosa samples were provided by the National Academy of Agricultural Science, Rural Development Administration, Wanju, Jeonbuk, Republic of Korea. A sample (CNUINS 201603) has been preserved as specimen in the archive of Pharmacognosy Laboratory of Chungnam National University .2.2.1N-acetyldopamine dimers, i.e. -2--3-acetylamino-7-(N-acetyl-2″-aminoethyl)-1,4-benzodioxane (DAB1) and -2--3-acetylamino-7-(N-acetyl-2″-aminethylene)-1,4-benzodioxane (DAB2), as bioactive compounds were isolated and purified from O. chinensis sinuosa3/MeOH  to produce six fractions of A-F. Fraction D (35.7 g) was further separated using silica gel vacuum liquid chromatography using a gradient mobile system of n-hexane/EtOAc  and CHCl3/MeOH  to yield 6 sub-fractions of D1-D6. Fraction D5 (18.3 g) was chromatographed on reversed phase medium pressure liquid chromatography using a SNAP Cartridge KP-C18-HS, (340 g) and eluted with a gradient solvent system acetonitrile (MeCN)/H2O (0.1:0.9 → 0.6:0.4) to afford six fractions of D5-1-D5-6. Compound 1 (389.2 mg) and compound 2 (37.2 mg) were isolated from fraction D5-2 (1.9 g) by preparative high-performance liquid chromatography using a Phenomenex Kinetex C18 column  and a mobile phase of MeCN/H2O (0.2:0.8 → 0.25:0.75)].Two 2.3’s Modified Eagles Medium  supplemented with 10% fetal calf serum (Invitrogen), and 1% penicillin–streptomycin with an atmosphere of 5% CO2 at 37 °C incubator.Murine macrophage cell line RAW 264.7 was procured from American Type Culture Collection  and cultured in Dulbecco2.44 cells/well were seeded in a 96-well culture plate and incubated for 24 h at 37 °C in a 5% CO2 incubator. Cells were further treated with DAB1 or DAB2 (0–500 µM) for 24 h, followed by the addition of MTT dye (5 mg/ml) for 4 h. Finally, the formed formazan crystals were re-dissolved in dimethyl sulfoxide, and sample absorbance was recorded at 540 nm.Effects of DAB1 and DAB2 on the viability of RAW 264.7 cells were examined using the 3--2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 1 × 102.55 cells/well) were grown in a 6-well culture plate for 24 h and treated with DAB1 or DAB2 (0–250 µM) for 1 h, followed by the supplement of LPS (1 μg/ml) for 24 h. Finally, the cell-free supernatant was collected and processed to quantify NO by mixing 150 µl of cell-free supernatant with 100 µl of Griess reagent. After 30 min of incubation, absorbance at 540 nm was recorded and results were expressed as NO%.NO was quantified calorimetrically using the Griess reagent. RAW 264.7 cells (1 × 102.65 cells/well) for 24 h. Cells were treated with DAB1 (250 µM) for 1 h, followed by exposure to LPS (1 µg/ml) for 12 h. After treatment, protein lysate was obtained using RIPA lysis buffer . Finally, protein content was quantified and equal amount of protein (30 µg) was loaded in SDS-PAGE (10%) and processed for Western blots, as described by Khaket et al. RAW 264.7 cells were grown in 6-well culture plate  was used as per the manufacturer's instructions for obtaining subcellular fractions (cytoplasmic and nuclear).2.85 cells/well) were grown in a 6-well culture plate and treated with DAB1 (0–250 µM) for 1 h, followed by the treatment of LPS (1 μg/ml) for 12 h. Cell free culture media was collected and centrifuged at 10,000×g for 5 min. Supernatant was collected and processed for the quantification of TNF-α and IL-1β using ELISA kit  according to manufacture instructions. Prostaglandin E2 (PGE2) was quantified using a commercial competitive ELISA kit  according to the manufacturer’s instructions.RAW 264.7 cells (1 × 102.94 cells/well) and then treated with DAB1  together with LPS (1 µg/ml) for 12 h. Finally, cells were washed with phosphate buffered saline and lysed using CelLytic™ M Reagent . The cell lysate was centrifuged at 10,000×g for 5 min in cooling conditions. The supernatant was collected and used as a source of lysate extract (crude enzyme). Enzyme activity was determined colorimetrically as previously described 3COONa, 10 mM NaCl, 1 mM DTT, and 1 mM EDTA, pH 5.5) and incubated for 10 min at 37 °C. Subsequently, 40 µM H-Gly-Phe-β naphthylamide was added. After 30 min incubation at 37 °C, 100 µl of sodium acetate buffer and 50 µl of coupling regent (0.1% Fast Garnet GBC in water) were added. The pink colored product was separated from the reaction mixture by partitioning with butanol, and the absorbance at 520 nm was recorded. The specific activity of cathepsin C was determined and expressed as percentage specific activity, considering only LPS treated control as 100%.RAW 264.7 cells were grown in a 24-well culture plate (1 × 102.10Enzyme inhibition activity was determined by mixing different concentrations of DAB1 (50–250 µM) with cathepsin C (10 ng/ml). The mixture was incubated at 37 °C for 15 min, followed by the addition of 40 µM substrate H-Gly-Phe-β naphthylamide. After 30 min incubation at 37 °C, 100 µl of sodium acetate buffer and 50 µl of coupling regent (0.1% Fast Garnet GBC in water) were added in the reaction mixture. The formed pink colored product was extracted with butanol and absorbance at 520 nm was recorded. Simultaneously, a control without inhibitor (DAB1) was prepared under the similar experimental conditions. Results were expressed as percent enzymatic activity, considering control as 100%.2.11m), maximal velocity (Vmax), and inhibition type. Additionally, Eadie-Hofstee plot was also constructed for the cathepsin C DAB1 inhibition by plotting a graph between velocity and velocity/substrate concentrations.Mode of inhibition was determined by caring out the enzyme reaction in presence and absence of inhibitor (DAB1) with varying amount of substrate. The enzyme cathepsin C (10 ng/ml) was incubated for 15 min with different concentrations of inhibitor , followed by the addition of varied amount of substrate (10–100 μM) in assay buffer. After 30 min incubation at 37 °C, the enzymatic activity at each substrate concentration was determined. Finally, a double reciprocal Lineweaver-Burk plot was constructed against substrate concentration and velocity to determine Michaleis-Menten constant (K2.122.12.1http://www.rcsb.org), and ligand (DAB1) three-dimensional structure was designed in the academic version of Maestro v12.3 . The co-crystallized cyanamide-based inhibitor compound 17 binding region Three-dimensional structure of cathepsin C (PDB ID: 3PDF) 2.12.2The molecular docking simulation for cathepsin C and compound DAB1 was performed using Chimera-AutoDock Vina plugin setup 2.12.3+ or Cl− ions. Later, system energy minimization was performed through nsteps = 50,000 through steepest descent method (1000 ps) and conjugated descent approach (100 ps). Moreover, the Particle Mesh Ewald approach was employed to calculate the energy as well as intermolecular interactions including van der Waals interactions. While a 14 Å distance limit was fixed for the short-range radius of van der Waals along with Coulomb cut-off and neighbor list were adjusted at a distance of 9 Å To validate the stability and to monitor the intermolecular interaction profiling with respect to time for the generated conformation of cathepsin C docked with potent ligand, molecular dynamics simulation was carried out for 100 ns using the Groningen Machine for Chemicals Simulations (GROMACS) 2018.1 package. The topology of the receptor was generated by using the Charmm 27 force field 2.12.4The essential dynamics, in terms of principal component analysis (PCA), which reflect the essential motions in protein required for enzymatic activity and residual displacement in the docked complex during molecular dynamic simulation with respect to time were calculated using Bio3d package 2.12.5http://rashmikumari.github.io/g_mmpbsa/accessed.21040920−3 kcal/mol) and T represent temperature (298.15 K).The major energy components responsible for cathepsin C-DAB1 complex stability were computed by the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method, as described by Kumari et al. 2.13p-value < 0.05.All the experiments were performed in triplicates, and results are presented as mean ± SD. SPSS-16 software was used to determine the statically difference between the groups employing one way ANOVA using the Duncan test for post hoc analysis at 33.1d4) δ 6.84–6.73 , 5.67 , 4.69 , 3.35 , 2.69 , 1.90 , 1.87 ; 13C NMR (150 MHz) δ 173.3 (CO), 173.2 (CO), 147.1 (C-4′), 146.5 (C-3′), 144.3 (C-8a), 142.2 (C-4a), 134.2 (C-7), 128.8 (C-1′), 123.2 (C-6), 120.6 (C-6′), 118.1 (C-8), 117.9 (C-5), 116.1 (C-5′), 115.6 (C-2′), 78.3 (C-2), 78.3 (C-3), 42.1 (C-2″), 35.8 (C-1″), 22.6 (CH3), 22.5 (CH3); electrospray ionization-mass spectrometry [ESI-MS] m/z 387 [M + H]+Compound 1 (DAB1): yellowish amorphous powder;d4) δ 7.30 , 6.90 , 6.86 , 6.85 , 6.81 , 6.76 , 6.74 , 6.10 , 5.69 , 4.71 , 2.03 , 1.87 , 13C NMR (150 MHz) δ 173.2 (CO), 170.6 (CO), 147.2 (C-4′), 146.5 (C-3′), 144.6 (C-8a), 142.6 (C-4a), 131.9 (C-7), 128.7 (C-1′), 122.9 (C-2″), 120.6 (C-6′), 120.3 (C-6), 118.3 (C-8), 116.1 (C-5), 115.6 (C-5′), 114.7 (C-2′), 114.0 (C-1″), 78.4 (C-2), 78.3 (C-3), 22.6 (CH3), 22.6 (CH3); ESIMS m/z 407 [M + Na]+.Compound 2 (DAB2): yellowish amorphous powder;3.2N-acetyldopamine derivative. Six aromatic protons at 6.84–6.73, two methines at 5.67 , 4.69 , two methylenes at 3.35  and 2.69 , and two acetyl groups at 1.90  and 1.87  were observed in the 1H NMR spectrum. The 13C NMR spectroscopic data revealed 20 carbon resonances assigned to two carbonyl groups , twelve aromatic carbons , two methines , two methylenes , and two acetyl groups . The planar structure was determined to be 2--3-acetylamino-7-(N-acetyl-2″-aminoethyl)-1,4-benzodioxane based on NMR spectroscopy analysis. The coupling constant between H-2 and H-3 was 7.11 Hz, indicating that it possesses trans H-2/H-3 stereochemistry. To determine the absolute stereochemistry, circular dichroism (CD) analysis was performed (N-acetyl-2″-aminoethyl)-1,4-benzodioxane  and nuclear magnetic resonance (NMR) techniques . The NMRerformed . The absodioxane a.Fig. 1C1H NMR and 13C NMR spectra of compound 2 (DAB2) were similar to those of compound 1 (DAB1), except two additional olefinic protons  and the absence of proton signals for the two methylene groups. The absolute configuration was determined to be 2R, 3S, based on the same Cotton effect as that of DAB1. Consequently, the structure of DAB2 was identified as -2--3-acetylamino-7-(N-acetyl-2″-aminethylene)-1,4-benzodioxane   b. A 1.7- < 0.05) a and b.FTogether with iNOS, higher expression of COX-2 has also been documented in various inflammatory diseases O. chinensis sinuosa against iNOS and COX-2 expression. However, the study done by Yoon et al. O. chinensis sinuosa extract on iNOS and COX-2 expression. It is conceivable that the crude extract contained DAB1 as one of the major components responsible for downregulation of iNOS and COX-2.So far, no literature suggests direct evidence of compounds isolated from 3.6p < 0.05) 1.5-fold reduced production of IL-1β was observed in macrophages treated with DAB1 and LPS, as compared to cells treated only with LPS (p < 0.05) 1.3-fold lower production of TNF-α was observed in macrophages treated with DAB1 and LPS, compared to only LPS stimulated macrophages  1.4-fold reduction of PGE2 level in the macrophages treated with DAB1 prior to the LPS compared to only LPS treated cells  1.5-fold lower nuclear translocation of NF-κB was observed in macrophages treated with LPS and DAB1, as compared to macrophages stimulated with LPS, suggesting a preventive role of DAB1 on NF-κB translocation and eventually inflammation  of 126.03 ± 5.04 µM  and 1.35-fold (7.76 ± 0.14 µM) lower, compared to the Km value obtained using 100 and 150 µM DAB1, respectively. The equilibrium constant of the inhibitor for the free enzyme  and enzyme-substrate complex  was derived from a secondary plot between inhibitor vs. slope and inhibitor vs. intercept, respectively  and 1.34-fold (8.29 µM) higher than the Km value observed for the enzyme treated with 100 and 150 µM of DAB1, respectively. The finding of the Eadie-Hofstee plot are comparable with the results obtained from the Lineweaver-Burk plot. These findings provided confirmatory evidence that DAB1 can inhibit cathepsin C. To the best of our knowledge, cathepsin C inhibition by isolate bioactive compounds O. chinensis sinuosa and its extract were not reported in the literature. Therefore, the current findings indicate the potential of DAB1 as a novel inhibitor that could be effective against cathepsin C mediated ailments, including inflammation.The cellular cathepsin C assay reported decreased activity of cathepsin C in the cells treated with DAB1. Decreased activity might be due to the effect of DAB1 on cathepsin C expression or by the direct interaction with an expressed product (cathepsin C), followed by blockage of the activity. To discern between the two possibilities, an  5.04 µM b. The inhibition . The Km ectively c. The inectively . The Km 3.10in vitro inhibitory results, molecular docking was performed  which together form a catalytic ternary complex in vitro data  noted for the receptor suggested stability of the protein d. This i3.12PCA was used to determine the domain dynamics motion in the docked receptor with ligand during 100 ns simulation and represented as fraction of variance (eigen fraction) with covariance matrix at 20 eigen model. The receptor cathepsin C is the monomer unit consisting of a single heavy chain, light chain and the exclusion unit. The DAB1 docked on the active pocket made by S1 unit of heavy chain and S2 unit of light chain. As depicted in Furthermore, the DAB1 induced conformational dynamic changes in cathepsin C was evaluated by DCCM analysis emphasizing on correlated motions during simulation at alpha carbon atom b. The DC3.13in vitro assay. These results were coherent with the predictions of docking scores and the enzyme inhibition assay, suggested the bioactive compound DAB1 as an inhibitor of cathepsin C.4O. chinensis sinuosa suppressed the expression of inflammatory mediators, iNOs and COX-2, including cytokines, via NF-κB signaling pathway in LPS induced macrophages. DAB1 effectively inhibited cathepsin C activity at the cellular level. Combined in vitro and in silico enzyme inhibition studies suggested a mixed-type inhibition. The cathepsin C inhibition and anti-inflammation behavior of DAB1 supports the notion of cathepsin C mediated anti-inflammatory activity via NF-κB signaling pathway. The collective results implicate DAB1 as a novel cathepsin C inhibitor that may be valuable as a preventive agent against inflammatory disorders after in vivo studies.DAB1 isolated from the Ashutosh Bahuguna: Methodology, Writing – original draft. Tejinder Pal Khaket: Methodology, Writing – original draft. Vivek K. Bajpai: Software. Shruti Shukla: Software. InWha Park: Validation, Visualization. MinKyun Na: Validation, Visualization. Yun Suk Huh: Data curation. Young-Kyu Han: Data curation. Sun Chul Kang: Methodology. Myunghee Kim: Conceptualization, Supervision, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


In the crystal, the molecules are linked by O—H⋯O and C—H⋯O hydrogen bonds into double ribbons. 19H21NO2, was developed with good yield and purity using the reaction of 4-hydroxy-3-methyl-2-butanone, benzaldehyde and ammonium acetate in glacial acetic acid as a solvent. The central piperidine ring adopts a chair conformation, and its least-squares basal plane forms dihedral angles of 85.71 (11) and 77.27 (11)° with the terminal aromatic rings. In the crystal, the molecules are linked by O—H⋯O and C—H⋯O hydrogen bonds into double ribbons. The Hirshfeld surface analysis shows that the most important contributions are from H⋯H (68%), C⋯H/H⋯C (19%) and O⋯H/H⋯O (12%) interactions.A new synthesis of the title compound, C Numerous biological effects including antiviral, antitumor, bactericidal, fungicidal and anti-inflammatory activities have been reported for these compounds -9-oxo-7-phenylethyl-6,8-diphenyl-3,7-diazabicyclo(3.3.1)nonane-1,5-dicarboxylate  to 1.6913 (blue) a.u. The large and small red spots indicate the O—H⋯O and C—H⋯O interactions. The two-dimensional fingerprint plots, shown in Fig. 4The Hirshfeld surface analysis of the title compound was performed using 2CO3 in a strongly basic reaction. After the extraction with diethyl ether of the by-product base (control of the completeness of extraction by TLC), the title compound was extracted with chloroform. After drying the chloroform extracts and distilling off the solvent, a white crystalline compound was obtained , readily soluble in chloroform, acetone, and hot ethanol  = 1.5Ueq(O). The C-bound H atoms were positioned geometrically  and refined using a riding model, with Uiso(H) = 1.5Ueq(C) and 1.2Ueq(C) for methyl and other H atoms, respectively.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021012640/yk2159sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021012640/yk2159Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021012640/yk2159Isup3.cmlSupporting information file. DOI: 2124980CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


We show that Vγ9Vδ2 T cells, expanded in vitro with zoledronic acid (Zometa or ZOL) and Interleukin-2 (IL-2), are efficient cancer cell killers with a trend towards increased killing efficacy after prolonged expansion time. Thus, Vγ9Vδ2 T cells expanded for 25 days in vitro killed prostate cancer cells more efficiently than Vγ9Vδ2 T cells expanded for 9 days. These data are supported by phenotype characteristics, showing increased expression of CD56 and NKG2D over time, reaching above 90% positive cells after 25 days of expansion. At the early stage of expansion, we demonstrate that Vγ9Vδ2 T cells are capable of cross-presenting tumor antigens. In this regard, our data show that Vγ9Vδ2 T cells can take up tumor-associated antigens (TAA) gp100, MART-1 and MAGE-A3 - either as long peptide or recombinant protein – and then present TAA-derived peptides on the cell surface in the context of HLA class I molecules, demonstrated by their recognition as targets by peptide-specific CD8 T cells. Importantly, we show that cross-presentation is impaired by the proteasome inhibitor lactacystin. In conclusion, our data indicate that Vγ9Vδ2 T cells are broadly tumor-specific killers with the additional ability to cross-present MHC class I-restricted peptides, thereby inducing or supporting tumor-specific αβTCR CD8 T cell responses. The dual functionality is dynamic during in vitro expansion, yet, both functions are of interest to explore in ACT for cancer therapy.The human Vγ9Vδ2 T cell is a unique cell type that holds great potential in immunotherapy of cancer. In particular, the therapeutic potential of this cell type in adoptive cell therapy (ACT) has gained interest. In this regard optimization of  Conventional T cells expressing αβ T cell receptors (TCR) have been characterized in detail with regards to antigen recognition, differentiation, and function . γδ T ceAn additional characteristic of Vγ9Vδ2 T cells is the capacity to cross-present antigen, i.e., to act as antigen presenting cells (APCs) . The terin vitro expanded TILs is associated with 50% objective and 20% complete responses  gradual freezing device. Cells were thawed in pre-warmed 37°C RPMI and counted after thawing using trypan blue staining and a microscope.Peripheral blood mononuclear cells (PBMC) from healthy donors were obtained from the blood bank at Rigshospitalet, Copenhagen, Denmark. Processing was completed within < 6 h for all sample specimens. PBMC were isolated by centrifugation with Lymphoprep™ (Axis-Shield PoC) (30 minutes at 1200 RPM) and cryopreserved at -150°C in fetal bovine serum (FBS) (GibcoBRL) + 10% dimethylsulfoxide (DMSO) (Sigma-Aldrich) using a CoolCellhttp://www.ebi.ac.uk/ipd/estdab/). All cancer cells were grown in RPMI 1640 GlutaMAX-I™ medium  supplemented with 10% FBS (R10). Prior to cytotoxicity assays, the cancer cells were left untreated or pre-treated with 10 µM ZOL for 24 h.Cancer cell lines A2058 (melanoma), MDA-MB-231 (breast cancer), PC-3 (prostate cancer), U266 (myeloma) and K562 (chronic myelogenous leukemia) were all purchased from the American Type Culture Collection (ATCC). The FM55-1  and FM86  cancer cell lines were obtained from European Searchable Tumor Cell Line and Data Bank (ESTDAB) . On day 2, 1000 U/ml IL-2 (Preprotech) was added, and every second or third day onwards, the cultures were supplemented with 1 ml fresh medium and 1000 U/ml IL-2. Purity of the Vγ9Vδ2 T cells was tested at day 9 by flow cytometry. We are aware that different groups use different amount of ZOL and IL-2. In our hands, we found expansion of Vγ9Vδ2 T cells to be most efficient when using 10µM ZOL, combined with either high IL-2 (1000U/ml) or low IL-2 + low IL-15 (100U/ml IL-2 + 100U/ml IL-15) (Vγ9Vδ2 T cells were cultured in X-vivo 15 medium (Lonza) supplemented with 5% human serum (X-vivo +5% HS) (Sigma-Aldrich). Vγ9Vδ2 T cells were expanded from thawed PBMC. On day zero, 1x10l IL-15) . We haveαβTCR T-cell culture specific for CMV (CMV short) was generated from healthy donor (HD220) by stimulating with irradiated CMV-peptide-loaded Vγ9Vδ2 T cells . The following day, 40 U/ml IL-7 and 20 U/ml IL-12 (PeproTech) were added. Stimulation of the cultures was carried out every 8 days with CMV-peptide–loaded irradiated autologous Vγ9Vδ2 T cells. The day after peptide stimulation, 120 U/ml IL-2 (PeproTech) was added. Specificity of the T cell culture was tested by chromium release assays and IFNγ ELISPOT.280-288-specific TCR 296 . Plasmids were linearized, purified , and in vitro transcribed 8 cells/ml in the same media. The cell suspension was pre-incubated for 5 min on ice and gp100 TCR encoding mRNA  was added to PBMCs before transfer to a 4-mm gap electroporation cuvette. Cells were pulsed using a BTX 830 square-wave electroporator , adjusted to a single pulse, 500 V, 5 ms. After electroporation, cells were transferred to pre-warmed culture medium and incubated in humidified atmosphere with 5% CO2. Mock-(H2O) transfected PBMCs were used as controls. Specificity of the T cell culture was tested by chromium release assays and IFNγ ELISPOT.T cells expressing αβTCR specific for gp100 (gp100 short) were generated by mRNA transfection. The coding sequence of the gp100 TCR 296  α and β TCRα and β sequences of the HLA-A2-restricted MART-1-specific A42 T cell clone  were cod6/ml per well in RPMI1640 supplemented with 10% human serum, 1% L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin  and 100 U/ml IL-2 (Cancernova). Then, cells were activated with 5 μg/ml OKT3  and 1 μg/ml anti-CD28 (BD Pharmingen) for 2 days.Transgenic TCR expression in T cells was achieved as described . Briefly®-LT1 Reagent (Mirus) according to the manufacturer’s protocol. Virus supernatant was harvested after 48 h and bound to RetroNectin®  coated plates by centrifugation.Amphotrophic TCR-A42-encoding retroviruses were generated as described  using TrPBMCs, which were activated for 2 days, were added to virus-coated plates for 24 h, then split to freshly virus-coated plates and cultivated for another 3 days. Transduced PBMCs were transferred to uncoated plates and cultivated for at least 12 additional days, reducing the amount of IL-2 to 50 U/ml. TCR-A42 surface expression was determined at day 12 after transduction using anti-mouse TCRß-constant region-Pacific Blue (BioLegend) and anti-human CD8α-V500 (BD Pharmingen) antibodies. Furthermore, specificity of the T cell culture was also tested by chromium release assays and IFNγ ELISPOT.a3a TCR . After 48 h, lentiviral supernatant was harvested. T cells were transduced by incubation with filtered lentiviral supernatant + 1000 U/ml rhIL-2 for 72 h before sorting using a FACSAria cell sorter .Lentivirus were produced and T cells transduced as previously described . In brie5 of the transduced and sorted T cells were put into a rapid expansion protocol (REP) consisting of feeder cells  at a ratio of 1:200 in T25 tissue culture flasks , 20 ml x-vivo medium + 5% human serum, 30 ng/ml anti-CD3  and 6000 U/ml rhIL-2. On day 14 of REP, T cells were harvested and routine assays for receptor expression via flow cytometry and mycoplasma were conducted. Furthermore, specificity of the T cell culture was tested by chromium release assays and IFNγ ELISPOT.1-2 x 10® software from ACEA biosciences.Flow cytometry was used to study expression of surface- and intracellular markers on cells. In short, cells were washed twice in FACS buffer (PBS +2%FCS) before and after staining. Antibodies were mixed to a total volume of 50 µl, and cells were stained for 30 minutes at 4°C. Vγ9Vδ2 cells were co-cultured with PC3 cells (cancer cells), either non-stimulated or stimulated with 10 µM ZOL, at a ratio (1:1). The cells were incubated for 5 hours in the presence of Brefeldin A (BioLegend). Addition of cell culture medium served as a negative control, while 5 ng/ml PMA (Sigma Aldrich) plus 75 nM Ionomycin (Sigma Aldrich) were used as a positive control. After incubation, cells were centrifuged and washed twice with PBS + 2% FBS. Staining with surface antibodies was performed as described above. Then cells were fixed and permeabilized as described in detail in the manual of the Intracellular Fixation & Permeabilization Buffer Set (eBioscience). In short, cells were fixed overnight at 4°C in 200 µl Fixation Buffer per well. After centrifugation, cells were washed twice with 150 µl Permeabilization Buffer per well. Staining with intracellular antibodies was then performed in the same manner as the surface antibody staining. Subsequently, cells were washed twice with Permeabilization buffer, resuspended in 150 µl PBS + 2% FBS and acquired on the NovoCyte Quanteon.6 T cells in 50 mL of 1x PBS, 0.5% bovine serum albumin  and 2 mM EDTA. After the tetramer staining, CD3-BV421 (Biolegend), in a volume of 50 µl, was added directly into the tetramer/cell mix and incubated for 20–30 minutes in the dark and on ice. The transduced MAGE-A3  tetramer positive T cells were sorted directly into a rapid expansion protocol (REP) for further culturing. All antibodies, buffers and procedures were kept under sterile conditions to ensure aseptic sorting of cells for further culturing.Sorting was used to purify αβTCR-specific cultures and was performed on the FACS Aria(BD bioscience). For sorting, the MAGE-A3 transduced T cell cultures were first stained with HLA-A1 tetramers for 30 minutes at 37°C. For that, 20 ng of PE- and APC-conjugated MAGE-A3 specific tetramers were added to1 x 106 PBMCs/20 ml medium (X-vivo + 5% HS) to which IL-2 and anti-CD3 was added. The REP mix was then ready for the tetramer sorted MAGE-A3 positive T cells to be added. From here, the cells were given fresh IL-2 (3000 U/ml) and medium (X-vivo + 5% HS) three times a week, until the γδ T cells started growing, allowing for further analysis.To expand the MAGE-A3 positive T cells cells into high cells number, the rapid expansion protocol (REP) was used. For this, a REP mix was generated which included: feeder cells, 6000 U/ml IL-2 (Proleukin) (Peprotech) and 0,6 µg/20 ml anti-CD3 (clone OKT3) . Detailed protocol was described elsewhere , 44, but51Cr-release assays for cell-mediated cytotoxicity was carried out as described elsewhere , an enzyme downstream of IPP within the mevalonate pathway, which leads to accumulations off IPP intracellular making them more prone to recognition by Vγ9Vδ2 T cells. This has been shown repeatedly and the potential clinical application is being tested .2). Its high-density electrode array, covering the bottom of E96 plates, allows this system to measure the variation in impedance throughout time. This measurement is then converted into a cell index, which can be translated into cytotoxicity.To measure the cytotoxicity of Vγ9Vδ2 T cells against cancer cell over an extended period, the xCELLigence system was used. This assay is composed of one station with an E96 plate , which is stored within a standard tissue culture incubator  and each target cell was plated out in E96 plates  and incubated for 6-24 h to promote adhesion and initial proliferation without reaching full confluency. Vγ9Vδ2 T cells were then added to each well, at a titrated 3:1, 1:1 effector-to-target ratio. The cell index was continuously measured for the next 24 h. To determine minimum impedance, 100 µl of 10% TritonX-100 (Sigma-Aldrich) was added to separate wells. Additionally, wells with Vγ9Vδ2 T cells alone were included to account for the effector cells’ contribution to the cell index. Data was analyzed with the immunotherapy module of the 185 xCELLigence RTCA Software Pro (ACEA Biosciences) as reported previously , 47.Several things were considered to rule out batch differences. All cytotox assays were conducted with Vγ9Vδ2 T cell cultures expanded from three to five different donors. Also, two different approaches were tested in cytotox assays with PC3 as a target: first the cytotox assays were conducted at the specific day of expansion, while the Vγ9Vδ2 T cells were ‘in culture’, meaning that cytotox was done on day 9, then waited 5 days to setup another cytotox on day 14, and again on day 25. The same days as the cytotox assay were performed, Vγ9Vδ2 T cells ‘in culture’ were also cryopreserved. The second approach was then to thaw cells, frozen at day 9, 14 and 25, and perform a cytotox assay in one setup. Both approaches showed similar results and representative data are shown in in vitro for 9-11 days prior to the APC assay. The setup for the APC assay was as following: Day 1, Vγ9Vδ2 T cells were stimulated with 1 µM ZOL and 100 U/mL IL-2. Day 2, Vγ9Vδ2 T cells were exposed to either a long peptide or protein and incubated 24 h to allow for cross-presentation of antigens. Day 3, Vγ9Vδ2 T cells were carefully washed twice with PBS to remove excess peptide or protein within the supernatant. Antigen cross-presentation was measured by IFNγ or TNFα release, using ELISPOT assay  was added to Vγ9Vδ2 T cells. In short, on ‘Day 2’ of the antigen cross-presentation assay: 50µM of Lactacystin was added to the Vγ9Vδ2 T cells. After two hours of incubation at 37°C, 5% COOverview of peptides and proteins used for the APC assay can be found in ELISPOT assay was used to measure cross-presentation of antigens by Vγ9Vδ2 T cells. In brief, ELISPOT plates  were coated ON with IFNγ capture antibody (Ab) (Mabtech) and afterwards blocked by X-vivo medium. Vγ9Vδ2 T cells (target cells) were placed in the ELISPOT plate (setup in triplicates) and short control peptides were added at 5 nM (see sequence above), with and without Teff. cells. The effector cells were either CMV, gp100, MART-1 or MAGE-A3 specific αβTCR T cells. The cells were then incubated ON, after which, the plates were washed off and secondary biotinylated Ab (Mabtech) was added. After 2 h incubation, unbound secondary antibody was washed off and streptavidin conjugated alkaline phosphatase (Mabtech) was added for 1 h. Finally, unbound conjugated enzyme was washed off and the assay developed by adding BCIP/NBT substrate (Mabtech). Developed ELISPOT plates were analyzed on CTL ImmunoSpot S6 Ultimate-V analyzer using Immunospot software v5.1.Criteria for standard protocol guidelines as well as determination of ELISPOT responses have been a challenge. In this regard, these ELISPOT assays were conducted according to the guidelines provided by CIP . Signifi6 day-9 expanded Vγ9Vδ2 T cells were seeded in a round-bottom 96-well plate in X-vivo + 5% HS. Lactacystin (Sigma Aldrich) was dissolved in sterile H2O and added to the wells at final concentrations of 0 µM, 1 µM, 10 µM, 25 µM, 50 µM and 100 µM. After two hours of incubation at 37°C, 5% CO2, cells were washed twice with PBS + 2% FBS and stained with the following extracellular antibodies in a total volume of 50 µl for 20 min at 4°C: anti-CD3 PE-Cy7, anti-TCRγ/δ FITC and anti-HLA-ABC BV711. After two washes with PBS + 2% FBS, apoptotic and dead cells were marked by staining with the Pacific Blue™ Annexin V/SYTOX™ AADvanced™ Apoptosis Kit (Invitrogen™). Annexin V binds to phosphatidylserine exposed on the outer membrane of apoptotic cells and SYTOX™ AADvanced™ Dead Cell Stain detects necrotic cells due to their loss of membrane integrity. A stain master mix was prepared by diluting Pacific Blue™ Annexin V and SYTOX™ AADvanced™ in 1X Annexin binding buffer. Cells were stained with 100 µl stain master mix per well for 30 min at 4°C and acquired on a NovoCyte Quanteon (ACEA Biosciences) without further washes.To detect apoptotic and dead cells after incubation with proteasome inhibitor Lactacystin, 0.5 x 10http://www.rstudio.com/).Statistical analyses were conducted using Graph-Pad Prism 7 . Differences between groups were determined by a paired T test. ELISPOT responses were analyzed using distribution free resampling (DFR) method, described by Moodie et al. for statistical analysis of ELISPOT responses , 50. TheVγ9Vδ2 T cells ability to kill cancer cell lines of various origins, was tested in a 4 h chromium release assay. Expanded Vγ9Vδ2 T cells from 2-5 healthy donors were used as effector cells to kill target cells, with or without sensitization with ZOL :(T) target cell ratio of 1:1. Exemplified in Finally, we setup an ICS assay, to investigate the activation of Vγ9Vδ2 T cells upon tumor engagement. Vγ9Vδ2T cells were co-cultured with PC-3 with or without ZOL, for 5 hours, and expression of IFNγ, TNFα and IL-2 was measured  express co-stimulatory markers . To veri+ αβTCR T effector cells (Teff.) that recognized, gp100 or MART-1 in an HLA-A*02:01-restricted manner, or MAGE-A3 with HLA-A*01:01 restriction. These were generated by either transfection or transduction of the TCR into αβ T cells . The specificity of the three αβTCR T effector cells (Teff.) was established using IFNγ ELISPOT upon recognition of the minimal gp100, MART-1, or MAGE-A3 peptide  which contains the minimal epitope flanked on both sides by several amino acids Table 2;To further strengthen these results, we included a proteasome inhibitor, lactacystin. To ensure that lactacystin was not toxic to the cells, an apoptosis assay was performed , The Aase and Ejnar Danielsen Foundation, The Dagmar Marshalls Foundation, the A.P. Moller foundation, The Danish Council for Independent Research (DFF – 1331 – 00095B), and Dansk Kræftforskningsfond.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.