IIINa(3-OHben)4[12-MCMn(III)N(shi)-4](H2O)4·10DMA, 1, and DyIIINa(4-OHben)4[12-MCMn(III)N(shi)-4](H2O)4·4DMF, 2, where MC is metallacrown, shi3− is salicylhydroximate, 3-OHben is 3-hydroxybenzoate, DMA is N,N-dimethylacetamide, 4-OHben is 4-hydroxybenzoate, and DMF is N,N-dimethylformamide, consist of a macrocyclic molecule with an [MnIII—N—O] repeat unit. For both 1 and 2, a DyIII ion is captured on the convex side of the central cavity, while a Na+ ion is captured on the concave side of the cavity. Four 3-hydroxybenzoate or 4-hydroxybenzoate anions bridge between the ring MnIII ions and the central DyIII ion.The metallacrown complexes Dy 2-3-hydroxybenzoato)dysprosium(III)tetramanganese(III)sodium(I) N,N-dimethylacetamide decasolvate, [DyMn4Na(C7H5O3)4(C7H4NO2)4(H2O)4]·10C4H9NO or [DyIIINa(4-OHben)4{12-MCMn(III)N(shi)-4}(H2O)4]·10DMA, 1, and tetra-μ-aqua-tetrakis{2-[azanidylene(oxido)methyl]phenolato}tetrakis(μ2-3-hydroxybenzoato)dysprosium(III)tetramanganese(III)sodium(I) N,N-dimethylformamide tetrasolvate, [DyMn4Na(C7H5O3)4(C7H4NO2)4(H2O)4]·4C3H7NO or [DyIIINa(3-OHben)4{12-MCMn(III)N(shi)-4}(H2O)4]·4DMF, 2, and where MC is metallacrown, shi3− is salicylhydroximate, 3-OHben is 3-hydroxybenzoate, DMA is N,N-dimethylacetamide, 4-OHben is 4-hydroxybenzoate, and DMF is N,N-dimethylformamide, are reported. For both 1 and 2, the macrocyclic metallacrown consists of an [MnIII—N—O] ring repeat unit, and the domed metallacrown captures two ions in the central cavity: a DyIII ion on the convex side of the metallacrown and an Na+ ion the concave side. The MnIII ions are six-coordinate with an elongated tetragonally distorted octahedral geometry. Both the DyIII and Na+ ions are eight-coordinate. The DyIII ions possess a square-antiprismatic geometry, while the Na+ ions have a distorted biaugmented trigonal–prismatic geometry. Four 3-hydroxybenzoate or 4-hydroxybenzoate ligands bridge each MnIII ion to the central DyIII ion. For 1, whole-molecule disorder is observed for the main molecule, excluding only the DyIII and Na+ ions, and the occupancy ratio refined to 0.8018 (14):0.1982 (14). Three DMA molecules were refined as disordered with two in general positions by an approximate 180° rotation and the third disordered twice by general disorder as well as by an exact 180° rotation about a twofold axis that bisects it. The occupancy ratios refined to 0.496 (8):0.504 (8), 0.608 (9):0.392 (9), and 2×0.275 (7):2×0.225 (7), respectively. For 2, segments of the metallacrown are disordered including the DyIII ion, one of the Mn ions, two of the Mn-bound 4-hydroxybenzoate ligands, the Mn-bridging salicylhydroximate ligand, and portions of the remaining three shi3− ligands. The occupancy ratio for the metallacrown disorder refined to 0.849 (9):0.151 (9). Two DMF solvent molecules are also disordered, each over two orientations. The disorder ratios refined to 0.64 (3):0.36 (3) and to 0.51 (2):0.49 (2), respectively. For 2, the crystal under investigation was refined as a non-merohedric twin by a 90° rotation around the real a axis [twin ratio 0.9182 (8):0.0818 (8)].The syntheses and crystal structures for the compounds tetra-μ-aqua-tetrakis{2-[azanidylene(oxido)methyl]phenolato}tetrakis(μ The self-assembly synthetic strategy of MCs lends itself to the ability to place metal ions in specific positions in the molecules and the controllable formation of specific molecules. While heterobimetallic MCs have been known since the 1990s, heterotrimetallic MCs have only been recently reported  were first discovered in 1989 by Pecoraro, and the compounds have grown into a class of coordination complexes with a wide range of applications including single-molecule magnets, magnetorefrigerants, luminescent agents, cell imaging agents, and magnetic resonance imaging agents (Mezei LnIIINa(OAc)4[12-MCMn(III)N(shi)-4](H2O)4 complexes, where LnIII is PrIII to YbIII (except PmIII) and YIII, −OAc is acetate, and shi3− is salicylhydroximate, that were the first heterotrimetallic MCs and the first 12-MC-4 complexes to bind a lanthanide ion in the central cavity 4[12-MCMn(III)N(shi)-4] complexes, where LnIII is YIII, ErIII, and DyIII, and X− is 2-hydroxybenzoate, benzoate, and trimethylacetate, which demonstrate that the bridging carboxylate anion can be easily substituted in these structures N(shi)-4] complexes with DyIII as the central lanthanide ion  version of the MCs behaves as an SMM, while the benzoate, acetate, and trimethylacetate analogues do not possess any SMM behavior. 2-Hydroxybenzoic acid has the smallest pKa value (2.98) of the species investigated, and the subsequent pKa values increase from benzoic acid (4.20) to acetic acid (4.76) to trimethylacetic acid (5.03). Thus, 2-hydroxybenzoate is the most electron-withdrawing of the set of anions, and this could affect the magnetic coupling between the ring MnIII ions and central DyIII ion.In 2014 we reported a series of IIINa(3-OHben)4[12-MCMn(III)N(shi)-4](H2O)4·10DMA, 1, and DyIIINa(4-OHben)4[12-MCMn(III)N(shi)-4](H2O)4·4DMF, 2, where 3-OHben is 3-hydroxybenzoate, DMA is N,N-dimethylacetamide, 4-OHben is 4-hydroxybenzoate, and DMF is N,N-dimethylformamide. The pKa values of 3-hydroxybenzoic acid and 4-hydroxybenzoic acid are 4.08 and 4.57, respectively, which are greater than the pKa of 2-hydroxybenzoic acid. Future studies will investigate the magnetic properties of 1 and 2 and the impact of the identity of the bridging ligand on the single-molecule magnetism of the MCs.Herein we report the syntheses and crystal structures of DyIIINa(3-OHben)4[12-MCMn(III)N(shi)-4](H2O)4·10DMA, 1, and DyIIINa(4-OHben)4[12-MCMn(III)N(shi)-4](H2O)4·4DMF, 2, both possess the typical 12-MC-4 framework with a repeat unit of MnIII–N–O that recurs four times to generate an approximately square-shaped molecule . Beyond overall molecular charge considerations, the metal oxidation states are confirmed by average bond lengths and bond-valence sum (BVS) values :0.1982 (14). For 2, large sections of the metallacrown are disordered, including the DyIII ion, Mn1, two of the 4-hydroxybenzoate ligands bound to Mn1 and Mn2, the shi3− ligand that connects Mn1 and Mn4, and portions of the remaining three shi3− ligands. The occupancy ratio for the metallacrown disorder refined to 0.849 (9):0.151 (9). Complete details describing the treatment of the disorder are given in the Refinement section. The following structural descriptions focus only on the major disorder components.The metallacrown complexes Dye Figs. 1 and 2 ▸.1 and 2, each MnIII ion is six-coordinate, with a tetragonally distorted octahedral geometry. The elongated Jahn–Teller axis along the z direction is expected for a high-spin d4 electron configuration. The geometry assignment is supported by a continuous shape measures (CShM) analysis  Å for 1 and 1.076 (7) Å for 2] than to the mean plane of oxime oxygen atoms [1.546 (3) Å for 1 and 1.593 (7) Å for 2], indicating that the geometry is distorted from an ideal square antiprism geometry. The mean plane distances were calculated with SHELXL2018/3 ; however, a square-antiprismatic geometry assignment is comparable . Both values are above 3.0; thus, there are substantial distortions from each ideal geometry. The biaugmented trigonal–prismatic geometry is a trigonal prism capped on two of the three rectangular faces. As for the DyIII ion, the Na+ ion is surrounded by two groups of oxygen atoms. One group of oxygen atoms is formed from the oxime oxygen atoms of the MC cavity, and the second group is comprised of four oxygen atoms from water molecules. The Na+ ion is positioned closer to the mean plane of water oxygen atoms [0.677 (5) Å for 1 and 0.561 (9) Å for 2] than to the mean plane of the oxime oxygen atoms [1.922 (4) Å for 1 and 1.991 (9) Å for 2].The Nas Table 3. The CSh1 and 2 solvent molecules are located in the structure, which are also hydrogen bonded to their respective MCs (described in the Supramolecular features section). For 1, the DMA molecules associated with N2 and N3 are disordered over two positions with occupancy ratios that refined to 0.496 (8):0.504 (8) and 0.608 (9):0.392 (9), respectively. The DMA molecule associated with N4 is disordered over four positions with occupancy ratios that refined to 2×0.275 (7):2×0.225 (7). For 2, two DMF molecules associated with N6 and N7 are not disordered, while the two DMF molecules associated with N5 and N8 are disordered over two different orientations, which refined to 0.64 (3):0.36 (3) and 0.51 (2):0.49 (2), respectively. Complete details describing the treatment of the solvent disorder are given in the Refinement section.Lastly, in both 1 and 2 the solvent molecules form hydrogen bonds with the MC complexes. For 1, the MC complex forms hydrogen bonds to the DMA molecules, and the MCs are interconnected via the DMA molecules (Table 4i) of a DMA molecule . In addition, the water molecule (O7) coordinated to the central Na+ ion hydrogen bonds to the carbonyl oxygen atoms (O8 and O8ii) of two DMA molecules . Then, the methyl group (associated with C17) of the same DMA molecules forms a C—H⋯O interaction with the hydroxyl group (O6iii) of a 3-hydroxybenzoate of a neighboring MC . These interactions are repeated about the fourfold axis of the MC; thus, a network is generated between neighboring MCs mediated by the DMA molecule associated with N2. The connection between the neighboring MCs, the hydrogen bonds between the MCs and the DMA molecules, and pure van der Waals forces contribute to the overall packing of the molecules.For both s Table 4. The hyd2, several DMF molecules are hydrogen bonded to each metallacrown and a small hydrogen-bonding network exists between neighboring metallacrowns  of the metallacrown . In addition, several hydrogen bonds exist between neighboring metallacrowns  of a 4-hydroxybenzoate anion forms a hydrogen bond to O3i (a phenolate oxygen atom of a shi3− ligand) of a neighboring MC through two hydrogen bonds: O15—H15O⋯O3i and C32—H32⋯O3i  complexes have been previously reported. Four of the metallacrowns contain both DyIII and Na+ ions in the central cavity of the MC. The complexes have different bridging carboxylate anions – acetate (OAc), benzoate (ben), 2-hydroxybenzoate (2-OHben), and trimethylacetate (TMA): Dy(OAc)4Na[12-MCMn(III)N(shi)-4](H2O)4·6DMF, 3 N(shi)-4](H2O)4·5DMF, 4 N(shi)-4](DMF)(H2O)3·4DMF, 5 N(shi)-4](H2O)2.59(DMF)1.41·4DMF·0.59H2O, 6 4K[12-MCMn(III)N(shi)-4](DMF)4·DMF N(shi)-4](H2O)4·4DMF·1.6H2O 0.5K[12-MCMn(III)N(shi)-4](DMF)1.5(H2O)3.5·5DMF 3]{Dy(OAc)4[12-MCMn(III)N(shi)-4]} N(shi)-4] complexes 3–6 that also capture a dysprosium and a sodium cation in the central cavity. The use of 3-hydroxybenzoate and 4-hydroxybenzoate does not significantly alter the overall MC framework as a structural comparison of complexes 1–6 reveals that the metrical parameters of the structures are similar  and dysprosium(III) nitrate pentahydrate (99.9%) were purchased from Alfa Aesar. Manganese(II) acetate tetrahydrate (99+%) was purchased from Acros Organics. N,N-dimethylformamide (ACS grade) and methanol (ACS grade) were purchased from Pharmco–Aaper. N,N-dimethylacetamide (>99.5%) was purchased from VWR Chemicals BDH. All reagents were used as received and without further purification.Sodium 3-hydroxybenzoate (>99.0%) and sodium 4-hydroxybenzoate (>99.0%) were purchased from TCI America. Salicylhydroxamic acid (HIIINa(3-OHben)4[12-MCMn(III)N(shi)-4](H2O)4·10DMA, 1.Synthesis of Dy Manganese(II) acetate tetrahydrate  was dissolved in 8 mL of DMA, resulting in a clear orange solution. In a separate beaker, dysprosium(III) nitrate pentahydrate  and salicylhydroxamic acid  were dissolved in 8 mL of DMA, resulting in a clear and colorless solution. In another beaker, sodium 3-hydroxybenzoate  was mixed in 8 mL of DMA, resulting in an opaque yellow mixture as not all of the reagent dissolved. Then the manganese(II) acetate solution was added to the Dy(NO3)3/H3shi solution, resulting in a dark-brown solution. Following, the sodium 3-hydroxybenzoate solution was added to the former solution and no color change was observed. The solution was stirred overnight and filtered the next day. A brown precipitate and clear and colorless solid were recovered and discarded. The filtrate was a dark-brown solution. Slow evaporation of the filtrate at room temperature afforded X-ray quality black/dark-brown block-shaped crystals after six days. The percentage yield was 44% based on dysprosium(III) nitrate pentahydrate.IIINa(4-OHben)4[12-MCMn(III)N(shi)-4](H2O)4·4DMF, 2.Synthesis of Dy Manganese(II) acetate tetrahydrate  was dissolved in a solvent mixture of 5 mL of DMF and 5 mL of methanol, resulting in a clear orange solution. In a separate beaker, dysprosium(III) nitrate pentahydrate , sodium 4-hydroxybenzoate , and salicylhydroxamic acid  were mixed in a solvent mixture of 5 mL of DMF and 5 mL of methanol, and the resulting mixture had an opaque white color as not all of the reagents had dissolved. Then the manganese(II) acetate solution was added to the latter mixture, resulting in an opaque green solution. The solution was stirred overnight and filtered the next day. A green precipitate was recovered and discarded. The filtrate was a dark green–brown solution. Slow evaporation of the filtrate at room temperature afforded X-ray quality black/dark-brown block-shaped crystals after three weeks. The percentage yield was 56% based on dysprosium(III) nitrate pentahydrate.1, whole molecule disorder is observed for the main molecule, excluding only the Dy and Na ions. Equivalent disordered organic moieties were restrained to have similar geometries (SAME command of SHELXL), and ijU components of ADPs for all disordered atoms closer to each other than 2.0 Å were restrained to be similar (SIMU command of SHELXL). Subject to these conditions, the occupancy ratio refined to 0.8018 (14):0.1982 (14). Three DMA molecules were refined as disordered. The two DMA molecules associated with N2 and N3 are in general positions by an approximate 180° rotation. The third DMA molecule associated with N4 is disordered by an exact 180° rotation from a twofold axis that bisects it as well as by additional general disorder. All DMA moieties were restrained to have similar geometries (SAME command of SHELXL). All N—CH3 bond lengths were restrained to be similar in length and all 1,3 distances of the C—N—CH3 angles were also restrained to be similar to each other. ijU components of ADPs for all DMA atoms closer to each other than 2.0 Å were restrained to be similar, and the atoms of the fourfold-disordered molecule were restrained to be close to isotropic. The lowest occupancy DMA molecule (the minor component disordered by twofold symmetry) was restrained to be close to planar. Subject to these conditions the occupancy ratios of the DMA molecules associated with N2, N3, and N4 refined to 0.496 (8):0.504 (8), 0.608 (9):0.392 (9), and 2×0.275 (7):2×0.225 (7), respectively. Initially alcohol hydrogen atoms were allowed to rotate about their respective oxygen atoms, and water hydrogen-atom positions were refined while a damping factor was applied, and O—H and H⋯H distances were restrained to 0.84 (2) and 1.36 (2) Å, respectively. Some water hydrogen-atom positions were further restrained based on hydrogen-bonding considerations. In the final refinement cycles these hydrogen atoms were set to ride on their carrier oxygen atoms and the damping factor was removed. Additional crystal data, data collection, and structure refinement details are summarized in Table 7For 2 the crystal under investigation was found to be a non-merohedric twin. The orientation matrices for the two components were identified using the program CELL_NOW . Two solvate DMF molecules are disordered over different orientations. The major and minor disordered moieties were each restrained to have similar geometries. ijU components of ADPs for all disordered atoms closer to each other than 2.0 Å were restrained to be similar. Subject to these conditions the occupancy ratio for the main molecule disorder refined to 0.849 (9):0.151 (9). The disorder of the two DMF moieties refined to 0.64 (3):0.36 (3) for the DMF associated with N5 and to 0.51 (2):0.49 (2) for the DMF molecule associated with N8. Water hydrogen atom positions were refined and O—H and H⋯H distances were restrained to 0.84 (2) and 1.36 (2) Å, respectively. Some water hydrogen-atom positions were further restrained based on hydrogen-bonding considerations and were restrained to be at least 3.10 (2) Å from the sodium ion. Additional crystal data, data collection, and structure refinement details are summarized in Table 7The structure was solved by direct methods with only the non-overlapping reflections of component 1. The structure was refined using all reflections of component 1 (including overlaps), resulting in a minor-component fraction of 0.0818 (8). The 10.1107/S2056989020008853/pk2637sup1.cifCrystal structure: contains datablock(s) 2, 1, global. DOI: 10.1107/S2056989020008853/pk26371sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989020008853/pk26372sup3.hklStructure factors: contains datablock(s) 2. DOI: 2013185, 2013184CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H-1,3-benzodioxol-5-yl)-N-cyclohexylpenta-2,4-dienamide, (I), and 5--1-(pyrrolidin-1-yl)penta-2,4-dien-1-one (II), are derivatives of piperine, which is known as a pungent component of pepper. Their geometrical parameters are similar to those of the three polymorphs of piperine, which indicate conjugation of electrons over the length of the molecules. The extended structure of (I) features N—H⋯O amide hydrogen bonds, which generate C(4) [010] chains. The crystal of (II) features aromatic π–π stacking, as for two of three known piperine polymorphs.The title compounds, 5--N-cyclohexylpenta-2,4-dienamide, C18H21NO3 (I), and 5--1-(pyrrolidin-1-yl)penta-2,4-dien-1-one C16H17NO3 (II), are derivatives of piperine, which is known as a pungent component of pepper. Their geometrical parameters are similar to those of the three polymorphs of piperine, which indicate conjugation of electrons over the length of the molecules. The extended structure of (I) features N—H⋯O amide hydrogen bonds, which generate C(4) [010] chains. The crystal of (II) features aromatic π–π stacking, as for two of three known piperine polymorphs.The title compounds, 5-(2 E,4E)-1-piperidine, C17H19NO3, is the major pungent ingredient of Piperaceae pepper (Piper nigrum). Piperine is an amide having a methylenedioxyphenyl grouping as a characteristic of its chemical structure  and the cyclic polysaccharide cyclodextrin -5--N-cyclohexylpenta-2,4-dienamide, C18H21NO3, (I)E,4E)-5--1-(pyrrolidin-1-yl)penta-2,4-dien-1-one, C16H17NO3, (II)P of (I)etc.) will help to evaluate the inclusion behaviour of piperine with cyclodextrin.Therefore, the aim of this study was to synthesize the title compounds  and subtends a dihedral angle of 21.57 (4)° with the cyclohexyl ring. The bond distances and angles  of (I)et al., 2015Compound (I)Pbca with eight molecules per unit cell. The C13–C16/N1 ring is well described as being twisted with C14 and C15 deviating from C13/N1/C16 by 0.205 (2) and −0.382 (2) Å, respectively. The C9/O2/C10/O3/C11 ring has a clear tendency towards an envelope conformation [deviation of C10 from the other four atoms = −0.216 (2) Å]. The dihedral angle between the C13–C16/N1 and C6–C12/O2/O3 rings  is 12.29 (10)°. As with (I)Compound (II)Thus, we may conclude that the title compounds show intramolecular resonance from the amide group to the ether O atoms of the methylenedioxyphenyl moiety, similar to piperine.et al., 2016) refcode: PIPINE10] and form II (PIPINE12) in space group P21/n and form III (PIPINE13) in space group C2/c  chains ns Fig. 4 with adjThe structure of (II)et al., 2015M HCl to pH < 1. The resultant pale-brown precipitate was collected by filtration, washed with cold water and recrystallized from methanol solution to give piperic acid. The piperic acid (1.0 mmol) was dissolved in CH2Cl2 (5 ml) and oxalyl chloride (10 mmol) was added and the mixture was stirred at room temperature for 3 h. The solvent and excess oxalyl chloride were then evaporated under reduced pressure.Piperine was purchased from Fujifilm Wako Pure Chemical Co., Ltd. The synthesis of piperine derivatives was performed using a previously reported procedure (Takao 2Cl2 (2 ml) and cyclohexylamine (1.2 mmol) and Et3N (8 mmol) were added, and the mixture was stirred at 273 K for 5 h. Ice-cold water was added to the mixture, followed by extraction with chloroform (5 ml). The organic layer was dried over Na2SO4 and the solvent was evaporated under reduced pressure. The residue was purified by silica-gel column chromatography (eluent hexane:etyl acetate 1:1 v/v) to give (I)To prepare (I)Compound (II)Uiso(H) = 1.2Ueq(C). The hydrogen atom attached to N1 in (I)Uiso(H) = 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020004648/hb7897sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989020004648/hb7897Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020004648/hb7897IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989020004648/hb7897Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020004648/hb7897IIsup5.cmlSupporting information file. DOI: 1994582, 1994581CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the absence of donor γδT cells, IFN-γ production by CD8+ T cells was severely impaired. Vγ4 subset was the major γδT cell subset mediating the GVL effect in vivo, which was partially dependent on IL-17A. Meanwhile, donor γδT cells could mitigate acute GVHD in a murine allo-HSCT model by suppressing CD4+ T cell activation and the major γδT cell subset that exerted this protective function was also Vγ4 γδT cells. Therefore, our findings provide evidence that donor γδT cells, especially Vγ4 subset, can enhance GVL effect and mitigate aGVHD during allo-HSCT.Disease relapse and graft-versus-host disease (GVHD) are the major complications affecting the outcomes of allogeneic hematopoietic stem cell transplantation . While the functions of αβT cells are extensively studied, the role of donor γδT cells in allo-HSCT is less well defined. Using TCRδ Allogeneic hematopoietic stem cell transplantation  is one of the most curative options for treating leukemia and other hematopoietic malignant diseases , 2. But via major histocompatibility complex (MHC). The ligands of γδTCR include MHC-related and MHC-unrelated molecules. It is not clear which endogenous ligands activate γδT cells in most disease conditions. γδT cells also exhibit similar recognition mechanisms as NK cells. They can express NKG2D and KIRs, and recognize target cells expressing stress-induced ligands  and BALB/c (H2Kd) mice (aged 6–8 weeks) were purchased from Shanghai Laboratory Animal Center  and In Vivos (Singapore). CD45.1-C57BL/6 (H2Kb) mice were obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd  and In Vivos (Singapore). TCR-δ-/–C57BL/6 (H2Kb) mice were provided by Prof. Zhinan Yin . TCR-β-/–C57BL/6 (H2Kb) mice were purchased from The Jackson Laboratory . TCR-β-/–CD45.1-C57BL/6 (H2Kb) mice were generated by crossing CD45.1 with TCR-β-/-. IL-17A−/−-C57BL/6 mice were provided by Dr. Chen Dong . All mice are female and maintained in specific pathogen-free conditions and in accordance with the guidelines approved by the Institutional Laboratory Animal Care and Use Committee of Soochow University and National University of Singapore.Specific pathogen free C57BL/6 (H2Kd) lymphoma cell line was purchased from American Type Culture Collection . Luciferase-expressing A20 cells were generated by a lentiviral system. Briefly, the luciferase gene was ligated into a lentiviral plasmid . The luciferase-expressing lentivirus generated from 293T cells  was used to infect A20 cells to generate the A20-luc+/yfp cells. Stable luciferase-expressing A20 cells were sorted by flow cytometry , and cultured with RPMI 1640 supplemented with 10% FBS .A20  from a 137Cs source. Three hours later, 1×107 BMCs plus 5×106 splenocytes from WT or TCR-δ -/- C57BL/6 mice were intravenously injected into lethally irradiated BALB/c recipient. The survivals were observed, and the body weights and clinical scores were assessed every two or three days. The severity of aGVHD was assessed with a clinical GVHD scoring system as described by Cooke et al. in a blinded fashion. The degree of systemic GVHD was assessed by the sum of changes in five clinical parameters: weight loss, posture (hunching), activity, fur texture, and skin integrity  from a ntegrity .-/- mice were seeded into the antibody-coated plates (2.5 × 106 cells/ml) and cultured for seven days in the presence of rhIL-2 . The purity of expanded γδT cells was more than 95%. 1×107 γδT/Vγ1/Vγ4 cells were adoptively transferred into the recipient mice on day 0 of allo-HSCT. The purified anti-mouse TCRγδ antibody was purchased from BD Bioscience . The purified anti-mouse Vγ1 (2.11) and Vγ4 (UC3) antibodies were purchased from Sungene Bio-technological company .Firstly, 10 µg/ml anti-mouse TCRγδ (GL3), Vγ1 (2.11) or Vγ4 (UC3) antibody was coated in 6-well plates (1 ml/well) overnight at 4°C. The coated plates were washed gently three times with PBS. The splenocytes from CD45.1-TCR-βTo deplete Vγ1 or Vγ4 subset, polyclonal hamster IgG, purified anti-mouse Vγ1 (2.11) or purified anti-mouse Vγ4 (UC3) antibody  was intraperitoneally injected into the recipient mice (100 µg/200 µl/mouse) once weekly. The depletion efficiency was confirmed by flow cytometry , mice were anesthetized by injecting i.p. 10% chloral hydrate then injected i.p. with 100 μl of 150 μg/ml D-luciferin . Five minutes later, mice were imaged using Xenogen, IVIS 100 Bioluminescent Imaging System  to determine the level of leukemia burdens.5×10+T cells from the spleen of recipient mice were obtained and cocultured with A20 cells at different E:T ratios for 6 hours. The killing capability was assessed according to the manufacturer’s protocol. The percentage of cytotoxicity at each E:T ratio was calculated using the following formula: percentage of cytotoxicity = /(target maximum − target spontaneous) × 100%.Cytotoxicity assay was carried out using a cytotoxicity detection kit (LDH) . Expanded γδT/Vγ1/Vγ4 cells or isolated CD893), PE/APC-anti-mouse IL-17A (TC11-18H10.1), PE-anti-mouse-NKG2D (C7), FITC-antimouse-NKG2D (C7), PE-anti-mouse-IFN-γ (XMG1.2), PE/Cy7-anti-mouse TNF-α (MP6-XT22), FITC-anti-mouse-CD69(H1.2F3), PE-anti-mouse-CD62L(MEL-14), APC-anti-mouse-CD44 (BJ18) were purchased from Biolegend ; PE-Cy7-anti-mouse- Granzyme B (NGZB), APC-anti-mouse-Perforin (eBioOMAK-D) were purchased from eBioscience . FITC-anti-mouse-Vγ1, APC-anti-mouse-Vγ4 were purchased from Sungene Bio-technological Company . Flow cytometric analysis were performed using a FACS Canto II  or NovoCyte  flow cytometer and analyzed by the Flowjo software .Single cell suspensions from spleens, livers, lungs, and IELs were obtained according to the methods previously described  and analP-value <0.05 was considered statistically significant (*), the significance levels are marked as *p <0.05, ** p<0.01, *** p<0.001 and **** p<0.0001.One-way ANOVA was used to determine statistically significant differences among more than two experimental groups. The unpaired Student t-test was used to determine statistically significant differences between the two experimental groups. Data were analyzed using GraphPad Prism 5 software for Windows . 6 cells/mouse, iv.) from WT or γδT deficient (TCRδ-/-) C57BL/6 donor mice. A20 cells  were injected into the recipients intravenously on the day of transplantation  together with A20 cells   and BMCs from TCR-δ-/- mouse , as well as A20 cells  on day 0 of allo-HSCT  were administered once a week for 4 weeks. The depletion efficiency of either cell subset was confirmed by flow cytometry  derived from CD45.1-TCR-β-/- mice together with BMCs  from TCRδ-/- mice and A20 cells . CD8+ T cells from spleens of the recipient mice were isolated on day 7 post-transplantation and the cytotoxicity of CD8+ T cells against A20 cells was measured  and adoptively transferred them  into recipient mice of TCR-δ-/- BMCs  and A20 cells   and splenocytes  from WT or TCR-δ-/- mice (C57BL/6)   in the murine aGVHD model described before  in the WT recipients , National Natural Science Foundation of China , Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and start-up grant of National University of Singapore.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.


The crystal structure of the title compound is stabilized by the presence of weak C—H⋯N hydrogen bonds and slipped π–π interactions. Hirshfeld surface analysis showed that H⋯H contacts are the dominant interactions. a]benzimidazole fused-ring system in the title compound, C14H8N2OS2, is nearly planar, the r.m.s. deviation being 0.0073 Å. The thiazolo-benzimidazole-2-thione system is almost in the same plane as the furan-2-yl-methylene moiety, with a dihedral angle of 5.6 (2)° between the two least-squares planes. In the crystal, adjacent molecules are connected by weak intermolecular interactions (C—H⋯N and slipped π–π stacking) into a three-dimensional network. The nature of the intermolecular interactions was also quantified by Hirshfeld surface analysis. DFT analysis indicates a good agreement of the experimentally determined and the theoretically calculated molecular structures.The thiazolo[3,4- The furan-2-yl-methylene moiety is also planar, with an r.m.s deviation of 0.0028 Å for the six (C1–C5/O1) non H-atoms. The two ring systems are almost in the same plane, their least-squares planes subtending a dihedral angle of 5.6 (2)°. The molecule exists in a Z configuration with respect to the C5=C6 bond. The S1—C8 and S1—C6 distances, 1.739 (4) and 1.775 (3) Å, respectively, are in agreement with a C—S single bond of a thiazole ring . The presence of π–π stacking interactions is indicated by red and blue triangles on the shape-index surface as can be seen in Fig. 3b. In Fig. 3c, the other red spots indicate also the presence of a weaker C—H⋯S hydrogen bond (between H3 and S2) and C—H⋯N (between H1 and N2). The overall two-dimensional fingerprint (FP) plots, and those delineated into H⋯H, C⋯H/H⋯C, S⋯H/H⋯S, N⋯H/H⋯N and C⋯C contacts are shown in Fig. 4Hirshfeld surface (HS) analysis of the title compound was performed using Gaussian 09 . Frontier molecular orbitals and the molecular electrostatic potential were calculated using the same level of theory.The hybrid functional B3LYP  with the 6-311G  basis set  and LUMO , are plotted to specify the distribution of electronic densities. The electron distribution of the HOMO-1, HOMO, LUMO and the LUMO+1 energy levels are shown in Fig. 5et al., 2019et al., 2020Global chemical reactivity descriptor (GCRD) parameters can be obtained as reported in the literature  surfaces were computed at the B3LYP/6-311G  level with the optimized structure using a)benzimidazole (1) was obtained from amino phenylene dithiocarbamate and chloroacetic acid by the Hanztsch reaction. The title compound (3) was prepared by Knoevenagel condensation of furaldehyde 2  and the tricyclic compound  in acetic acid (10 ml) buffered by sodium acetate (0.02 mol). The reaction was monitored by TLC . After 4 h of refluxing and stirring, the brown solid obtained was filtered off, dried and recrystallized from ethanol to give the title compound, m.p. 493 K, in a yield of 85%.The synthetic route preparation of the title compound is illustrated in Fig. 71H NMR and 13C NMR) for (3). IR : 3099, 3076 and 3026 (Csp2—Harom), 1602 (C=N), 1555–1464 (C=C), 1390 (C=S), 1324 (–C—S–), 1259 (C—N) and 815, 759 (C—Haryl). 1H NMR  J (Hz): 6,6 , 6.80 , 7.45 , 7.60 , 7.70  , 7.80 , 8.50 . 13C NMR : 113.46, 113.51, 113.90, 117.15, 118.81, 120.65, 121.32, 125.59, 126.55, 131.00, 146.63, 149.48, 150.42 (C=N), 187.15 (C=S).Spectroscopic data  = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020015017/wm5587sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020015017/wm5587Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020015017/wm5587Isup3.molSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020015017/wm5587sup5.docxSelected bond lengths, bond angles and torsion angles for the non-hydrogen atoms, as determined by X-ray diffraction technique and DFT calculations. DOI: Click here for additional data file.10.1107/S2056989020015017/wm5587Isup5.cmlSupporting information file. DOI: 2043709CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The ligand is ambidentate and can coordinate via three nitrogen atoms or two sulfur and one nitrogen atoms. The cobalt(II) metal center has pseudo-octahedral geometry and based on the single-crystal structure, the pincer ligand coordinates in a meridional fashion with the metal and adjacent six-membered ring ligands all in a similar plane and forming two slightly distorted boat configurationsWe report here the synthesis, single-crystal structure, electrospray mass spectrum and NMR spectroscopy of a new six-coordinate cobalt(II) pincer complex. The pincer ligand, in this complex, which is novel, coordinates  2(C19H27N7S2)(H2O)], at 173 K has monoclinic (C2/c) symmetry. We report here the synthesis, single-crystal structure, electrospray mass spectrum and NMR spectroscopy of a new six-coordinate cobalt(II) pincer complex. The pincer ligand, in this complex, which is novel, coordinates via three nitrogen atoms (two triazole and one pyridine). The ligand is ambidentate and can coordinate via three nitrogen atoms or two sulfur and one nitrogen atoms. The cobalt(II) metal center has pseudo-octahedral geometry and based on the single-crystal structure, the pincer ligand coordinates in a meridional fashion with the metal and adjacent six-membered ring ligands all in a similar plane and forming two slightly distorted boat configurations. The other two coordinated monodentate ligands are one water molecule and two chloride ions with four cobalt(II) complexes in the unit cell. The asymmetric unit of the complex is comprised of half the pyridine ring and water molecule with the CoII atom at the center of the pincer situated about a twofold axis. The Co—N, Co—O, and Co—Cl bond lengths are consistent with single bonds. In the crystal, the complex forms a three-centre bifurcated weak hydrogen-bonding interaction with a chlorine ion, forming one intermolecular interaction with the pincer group and a water molecule and a second intramolecular interaction with a C—H group within the pincer group. Crystal packing is also highlighted with C22(6)>a<a infinite chains forming along [001] supported by R22(8)>a>a ring motifs, forming a three-dimensional supramolecular network structure. While some stacking of the pyridine rings in the unit cell is observed, there are no relevant π–π interactions in the crystal packing. The 1H and 13C{1H} NMR spectra of the complex are consistent with a plane of symmetry being present. The electrospray mass spectrum, which was collected in positive ion mode, showed the loss of one water molecule and one chloride ligand from the complex. In the future, we plan to screen this cobalt(II) complex for electrocatalysis reactivity.The structure of the title compound, [CoCl In this communication, we report the preparation, spectroscopic characterization, electrospray mass spectrometry, and single crystal structure of a cobalt(II) pincer complex that contains an ambidentate ligand  pincer complex that contained a pincer ligand precursor with methylene moieties connecting each triazole substituent to the pyridine in the pincer ligand precursor Fig. 2. We wondnd Fig. 2.19H29Cl2CoN7OS2, at 173 K whose structure has monoclinic (C2/c) symmetry . The ligand is ambidentate and can coordinate via three nitrogen atoms or two sulfur and one nitrogen atom. The cobalt(II) metal center has a pseudo-octahedral geometry and based on the single crystal structure, the pincer ligand coordinates in a meridional fashion with the metal and adjacent six-membered ring ligands all in a similar plane and forming two slightly distorted boat configurations pyridine diiodide methyl]pyridine was dissolved in 25 ml of 1,4 dioxane. To this solution 13.958 g (0.0759 mol) of iodobutane were added. This solution was heated at reflux for 18 h. After allowing the solution to cool, the mother liquor was decanted off. The precipitate was dissolved in minimal methanol and transferred to a round-bottom flask. The solvent was then removed under reduced pressure. Yield: 3.85 g (0.00748 mol) (59.4% yield).1H and 13C{1H} NMR spectroscopy.The product was characterized using 1H NMR : δ 10.32 , 9.30 , 7.97 , 7.52 , 5.75 , 4.32 , 1.85 , 1.32 , 0.92 . 13C {1H} NMR , δ 152.59 (pyridine C), 144.91 (triazole CH), 143.51 (triazole CH), 138.95 (pyridine CH), 122.85 (pyridine CH), 55.74 (CH2 linker), 47.42 (n-butyl CH2), 30.82 (n-butyl CH2), 18.79 (n-butyl CH2), 13.34 (n-butyl CH3).2,2′-bis  of 6 were dissolved in 15 mL of MeCN. To this solution, 0.800 g (0.0249 mol) of sulfur and 0.2162 g (0.001564 mol) of potassium carbonate were added. This mixture was heated at 398 K for 6 h in the microwave reactor. After the reaction was complete, the undissolved solids were removed by vacuum filtration and the remaining solvent was removed under reduced pressure. The product was purified by dissolving the product in CH2Cl2 and filtering it through an alumina column to removed undissolved sulfur. Mass product = 0.0934 g (2.24 x 10 −4 mol) (42.8% yield).1H and 13C{1H} NMR spectroscopy. The key feature of the NMR is that one of the acidic protons of the triazole, δ 10.32 in 6 was absent in the product and was presumably replaced with the thione moiety. There was also considerable shifting, to lower ppm values, of the aromatic, methylene and n-butyl CH2 proton resonances in 7 compared to the starting material 6.The product was characterized using 1H NMR : δ 8.65 , 7.75 , 7.02 , 5.41 , 3.98 , 1.72 , 1.29 , 0.92 . 13C {1H} NMR , δ 166.03 (C=S), 155.02 (pyridine C), 141.41 (triazole CH), 137.92 (pyridine CH), 120.23 (pyridine CH), 53.03 (CH2 linker), 45.14 (n-butyl CH2), 29.91 (n-butyl CH2), 19.04 (n-butyl CH2), 13.47 (n-butyl CH3).Aquadichloro---butyl]triazol-5-thione-1-yl)]pyridinecobalt(II)  and combined with 0.0333 g (4.43 × 10−4 moles) of potassium chloride (KCl) and dissolved in 10 mL of acetonitrile. The solution was refluxed for 20 h. The following day, the solution was filtered to remove undissolved material and the solvent was removed under reduced pressure. Yield: 0.162 grams (quantitative). Purple needle-shaped crystals suitable for X-ray diffraction were grown by a slow vapor diffusion of diethyl ether in to an acetonitrile solution containing the cobalt complex.19H29Cl2CoN7S2]·H2O:Analysis calculated for [C (583.46): C, 39.11; H, 5.36; N, 16.80. Found: C, 39.33; H, 5.07; N, 17.11.1H NMR  δ 8.61 , 7.71 , 6.91 , 5.37 , 3.95 , 1.70 , 1.26 , 0.88 . 13C {1H} NMR , δ 164.95 (C=S), 153.93 (pyridine C), 140.47 (triazole CH), 136.91 (pyridine CH), 119.19 (pyridine CH), 51.98 (CH2 linker), 44.13 (n-butyl CH2), 28.87 (n-butyl CH2), 18.01 (n-butyl CH2), 12.47 (n-butyl CH3).1H NMR and 13C{1H} spectrum of the complex was acquired in DMSO-d6. The NMR spectrum was consistent with the title complex. There was not considerable shifting of the proton resonances compared to the starting bis-thione precursor. In the 13C{1H} NMR spectrum, the C=S resonance shifted about δ 1 ppm to a lower chemical shift value.The 7 and 8 are given in Figs. S1 and S2, respectively, in the supporting information. In both 7 and 8 the supporting electrolyte is 0.2 M tetrabutylammonium tetrafluoroborate. The reference electrode is Ag wire, the working electrode is glassy carbon and the counter electrode is a platinum wire. The scan rate is 100 mV s-1.The cyclic voltammograms of Electronic absorption spectrum of 8 in acetonitrile (1.89 mM) : UV–Visible data: λ (nm), (∊ (M−1cm−1) (1.89 mM in MeCN) 234.00 (2480); 244.00 (2480); 368.00 (158); 574.00 nm (sh) (386); 589.00 (428); 682.00 (648).Uiso(H) = 1.2Ueq(C) or 1.5Ueq.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989020013547/jy2002sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020013547/jy2002Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020013547/jy2002sup2.pdfSupplementary figures. DOI: 2036378CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Page 1: Corine GeurtsvanKessel’s surname was misspelled as “Geurts-van Kessel” in the byline and was corrected on 6 November 2020.Volume 5, no. 6, e00941-20, 2020,


RUNX1) is required for definitive hematopoiesis; however, the functions of most human RUNX1 isoforms are unclear. In particular, the effects of RUNX1-205 (a novel splice variant that lacks exon 6 in comparison with RUNX1b) on human hematopoiesis are not clear. In this study, a human embryonic stem cell (hESC) line with inducible RUNX1-205 overexpression was established. Analyses of these cells revealed that induction of RUNX1-205 overexpression at early stage did not influence the induction of mesoderm but blocked the emergence of CD34+ cells, and the production of hematopoietic stem/progenitor cells was significantly reduced. In addition, the expression of hematopoiesis-related factors was downregulated. However, these effects were abolished when RUNX1-205 overexpression was induced after Day 6 in co-cultures of hESCs and AGM-S3 cells, indicating that the inhibitory effect occurred prior to generation of hemogenic endothelial cells, while the promotive effect could be observed during the late stage of hematopoiesis. This is very similar to that of RUNX1b. Interestingly, the mRNA expression profile of RUNX1-205 during hematopoiesis was distinct from that of RUNX1b, and the protein stability of RUNX1-205 was much higher than that of RUNX1b. Thus, the function of RUNX1-205 in normal and diseased models should be further explored.Runt-related transcription factor 1 ( RUNX1) is the key gene for human hematopoiesis, which plays a critical role in the development of hemogenic endothelium and hematopoietic stem cell (HSC) formation during embryogenesis (Runt-related transcription factor 1 (ogenesis . Embryonogenesis .RUNX1 gene is 216 kb long and located on human chromosome 21 (21q22.12) and has two promoters termed P1  and P2 . Seventeen transcripts of human RUNX1 have been identified, among which RUNX1a/b/c have been well studied , an mSin3A interaction domain (exon 6), and a transactivation domain (exons 7B and 8). Exon 6 is deleted in human RUNX1-205 in comparison with RUNX1b  analysis of co-cultured RUNX1-205/hESCs and the aorta-gonad-mesonephros (AGM-S3) cells revealed that treatment with DOX from Day 0 (D0) did not influence mesoderm induction . However, this effect was attenuated or abolished when DOX treatment was initiated after D6, and the hematopoiesis could even be stimulated in some degree with DOX treatment from D10 , and consequently, both CD34+CD43+ and CD34+CD45+ populations were lost. However, the production of CD34+ populations was almost normal when RUNX1-205 overexpression was induced from D6 or later and even significantly enhanced with RUNX1-205 induction from D10. This indicates that the development of hemogenic endothelium was blocked by induction of RUNX1-205 overexpression at early stage but not affected by RUNX1-205 induction after D6 (RUNX1-205 on hematopoiesis are similar to those of RUNX1b.The effects of from D10 . Generatafter D6 . These e+ population among green fluorescent protein positive (GFP+) cells was unaffected at D4 in co-cultures of RUNX1-205/hESCs and AGM-S3 cells treated with DOX from D0, indicating that RUNX1-205 overexpression did not block mesoderm induction , colony-forming unit-erythroid (CFU-E), colony-forming unit-mixed (CFU-Mix), and colony-forming unit-granulocyte/macrophage (CFU-GM) colonies (P < 0.05); however, the colony numbers were normal with RUNX1-205 induction from D6  co-cultured with AGMS-3 at D2 were further cultured in irradiated AGMS-3 with or without DOX . FACS analysis indicated that the production of CD34+CD43−, CD34+KDR−, and CD34+KDR+ populations significantly dropped after induction of RUNX1-205 overexpression from D2  did not affect the production of CD34+CD43+ and CD34+CD45+ populations, but promoted CD34−CD43+ and CD34−CD45+ populations (RUNX1-205 induction after the mesoderm−hemogenesis transition could not influence the development of hematopoiesis stem/progenitor cells (HSPCs) but promoted the production of CD34− blood cells at later stage , low at late stage (after D4), and upregulated at D14. By contrast, mRNA expression of RUNX1b was high at early stage (D2–D4), lowest at D6, and gradually increased at late stage (after D8). mRNA expression of RUNX1c was similar to that of RUNX1b but exhibited greater fluctuation  did not block induction of mesoderm, but blocked mesoderm–hemogenesis transition and the production of CD34+CD43− and its progenitor CD34+KDR+ cells  at early stage (+CD43+ and CD34+CD45+ populations at late stage. These effects were attenuated or even abolished by RUNX1-205 induction at late stage (D6) according to examination on co-culture cells or further hematopoiesis culture, which even indicated the promotion of hematopoietic cells in some degree  due to deletion of exon 6. This may mean that RUNX1b is more readily targeted for degradation via the ubiquitin–proteasome pathway, which would explain its lower stability  than at late stage (after D4), suggesting that this isoform mainly functions during mesoderm induction. By contrast, mRNA expression of RUNX1b was high at both early (D2–D4) and late (after D8) stages, indicating that this isoform plays important roles throughout hematopoiesis. mRNA expression of RUNX1-205 was much lower than that of RUNX1b even at early stage but was significantly higher than that of RUNX1c. This indicates that RUNX1-205 ought to be an important isoform related to hematopoiesis. Expression of RUNX1-205 and RUNX1b in co-cultures with AGM-S3 cells was high at early stage during generation of mesoderm, indicating that both isoforms are important at this stage, and decreased at the mesoderm−hemogenesis transition. Therefore, the ectopic expression of them at this point might block the transition from mesoderm to hematopoiesis. RUNX1b was also important at late stage of hematopoiesis, and consequently its expression gradually increased after D8. However, expression of RUNX1-205 remained low after D4 and was not upregulated until D14, which indicates that this isoform has a distinct role in comparison with RUNX1b at late stage.qRT-PCR analysis revealed that the mRNA expression profiles of RUNX1-205 showed a similar function to RUNX1b during hESC differentiation to hematopoiesis, detailed functions of RUNX1-205 in normal and diseased models still need to be further explored.Though Homo sapiens chromosome 21 and Mus musculus chromosome 16, were collected from GenBank. The following cDNA sequences were obtained using BLAST searches: chimpanzee NC_027889.1 (primate), house mouse NC_000082.6, zalophus NW_020884868.1, rhinoceros NW_004454184.1 , emu NW_020453926.1 (bird), crocodile NW_017728906.1 (reptile), African frog NC_030727.1 (amphibian), shark NW_018033230.1 , and spearfish NW_005819150.1 . Alignments were performed at https://www.genome.jp/tools-bin/clustalw and formatted at https://embnet.vital-it.ch/software/BOX_form.html.Genome sequences, including those of RUNX1-205 was synthesized, cloned into the PUC57 vector, excised using SnaBI/EcoRI, and inserted between the SwaI/EcoRI sites of PB-Tet-on-OE to yield the PiggyBac-based Tet-on inducible expression vector PB-Tet-on-GFP-T2A-hRUNX1-205. H1 hESCs (provided by Prof. Tao Cheng) were transfected with this vector using Lipofectamine 3000 (Invitrogen), selected by treatment with 1 μg/ml puromycin, and passaged using ReleSR (Stem Cell) to establish a hESC line with inducible RUNX1-205 overexpression . Induction of RUNX1-205 overexpression was confirmed by qRT-PCR and western blot analyses. Pluripotency was confirmed by western blot analysis of OCT4, SOX2, and NANOG.The coding region sequence of RUNX1b/hESCs and RUNX1-205/hESCs were dissected into small squares containing 0.5 × 103–1 × 103 cells, plated onto irradiated AGM-S3 cells in human pluripotent stem cell-maintaining medium . H1 hESCs were induced to undergo hematopoietic differentiation by co-culture with the mouse stromal cell-derived line AGM-S3 as reported previously . Brieflyg medium , cultureRUNX1b/hESCs and RUNX1-205/hESCs cultured in mTeSR1 were dissociated by treatment with 2 mg/ml dispase (Stem Cells), plated into 24-well ultra-low attachment plates (Corning), and grown in mTeSR1 containing 20 ng/ml bone morphogenetic protein 4 (BMP4) for 24 h . Thereafter, EBs were dispersed by pipetting and switched to mTeSR1 containing 20 ng/ml BMP4, 100 ng/ml stem cell factor (SCF), and 100 ng/ml Fms-related tyrosine kinase 3 ligand (FL) for 24 h (EB-D1). EBs were switched to EB culture media , 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 1% nonessential amino acids) at EB-D2. EBs were treated with 1 μg/ml DOX from EB-D0, EB-D2, EB-D4, or EB-D6, collected at EB-D10, and digested with 0.05% trypsin for further analysis.RUNX1b/hESCs or RUNX1-205/hESCs and AGM-S3 cells were treated with 1 μg/ml DOX (referred to as DOX-induced) from D0, D2, D4, D6, D8, or D10 and then dissociated at D4, D8, or D14 with 0.05%–0.25% trypsin/EDTA, which was inactivated by cold Dulbecco’s phosphate-buffered saline (D-PBS). Cells were pelleted, resuspended in 1 ml sorting medium (D-PBS containing 2% FBS), passed through a 70-μm membrane filter, blocked with rabbit serum on ice for 20 min, labeled with antibodies against CD34/KDR (at D4), CD34/CD43 (at D8), or CD34/CD45 (at D14), and analyzed by FACS . Protein samples were collected at 0 (0 day), 4, 8, 12, 24, 48 (2 days), and 96 h (4 days) and analyzed by western blotting.293T cells (ATCC) were transfected with 6–1 × 106 cells using 1 ml TRIzol (Life Technologies) and purified according to the manufacturer’s manual. Complementary DNA was synthesized using a reverse transcription kit (Bio-Rad). Each 20 μl reaction contained 4 μl 5× Mixture, 1 μl reverse transcriptase, 1 μg total RNA, and nuclease-free water to 20 μl. The conditions used for reverse transcription were as follows: 25°C for 5 min, 42°C for 30 min, 85°C for 5 min, and storage at 4°C. qPCR was performed using Fast Start Universal SYBR Green Master (Roche) on a CFX96™ real-time system (Bio-Rad). Each 15 μl reaction contained 7.5 μl 2× Mixture, 0.4 μl each primer (10 μM), 4.7 μl H2O, and 2 μl complementary DNA. The conditions were as follows: denaturation at 10 min at 95°C, followed by 45 cycles of 95°C for 15 sec, 58°C for 30 sec, and 72°C for 30 sec. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal control. The primers are listed in Total RNA was extracted from 0.5 × 104 co-cultured cells were added to 80% MethoCult H4230 (Stem Cell) containing 100 ng/ml SCF, 100 ng/ml interleukin-6 (IL-6), 10 ng/ml interleukin-3 (IL-3), 10 ng/ml FL, 10 ng/ml thrombopoietin (TPO), 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF), and 4 units/ml erythropoietin (EPO) and then incubated in 5% CO2 at 37°C for 14 days. The number of CFU-E colonies was calculated at 7–10 days, while the numbers of BFU-E, CFU-Mix, and CFU-GM colonies were calculated at 12–14 days as previously described . For morphological observation, cells were stained with MGG solution (MERCK) and then imaged using an Olympus BX53 microscope equipped with an oil objective.RUNX1-205/hESC co-cultures at D2 were dissociated with 0.05% trypsin solution and stained with anti-KDR antibody. KDR+ cells were sorted using a BD FACSJazz™ Cell Sorter, and their purity was confirmed by FACS. About 5 × 103 sorted cells were re-plated in 24-well plate on irradiated AGM-S3 for 10 days with or without DOX induction, refreshed with hematopoiesis-inducing medium every other day, and finally analyzed by FACS analysis.Noninduced or induced RUNX1-205/hESC co-cultures at D6 were dissociated with 0.05% trypsin solution and stained with anti-CD34/KDR antibody. Then CD34+KDR− cells were sorted, and ~5 × 103 cells were re-plated in 48-well plate with full-lineage hematopoietic differentiation (FLHD) medium  for 5 days treated with or without DOX, refreshed with media every day, and finally analyzed by FACS analysis.t-test. P < 0.05 was considered significant. Data were analyzed using FlowJo V10 and GraphPad Prism 5 software.All experimental data were described as mean ± SD. Statistical significance was evaluated using the Student’s Supplementary_material_mjaa019Click here for additional data file.


The X-ray crystallographic analysis shows that the silicon−silicon π single bond (2.853(1) Å) is far longer than the longest silicon−silicon bond so far reported. In spite of this unusually long bond length, the electrons of the 3p orbitals are paired, which is confirmed by measurement of electron paramagnetic resonance, and magnetic susceptibility and natural bond orbital analysis. The properties of the silicon−silicon π single bond are studied by UV/Vis and 29Si NMR spectroscopy, and theoretical calculations.A carbon–carbon double bond consists of a σ bond and a π bond. Recently, the concept of a π single bond, where a π bond is not accompanied by a σ bond, has been proposed in diradicals containing carbon and heteroatom radical centers. Here we report a closed-shell compound having a silicon–silicon π single bond. 1,2,2,3,4,4-Hexa- Examples of π single bonds in diradicals are rare, as they are typically unstable. Here, the authors describe a room temperature-stable closed-shell bicyclic tetrasilane that features a Si-Si π single bond between two of the non-adjacent Si centers. In this double bonding, two sp2 carbon atoms are faced in the direction of the sp2 orbital. Another possible bond is a π single bond, where two sp2 carbon atoms are faced in the bisecting direction between two sp2 orbitals ]2[P(i-Pr)2]2 was isolated as a stable singlet diradical and its structure and properties have been reported7.The concept of a carbon–carbon double bond is one of the fundamentals in organic chemistry. A carbon–carbon double bond is formed by overlapping of the als Fig. . This netert-butylbicyclo[1.1.0]-tetrasilane having a silicon–silicon π single bond was isolated as a closed-shell compound . The structural features and electronic properties of the silicon–silicon π single bond were revealed by X-ray crystallography, electron paramagnetic resonance (EPR), magnetic susceptibility, ultraviolet/visible (UV/Vis), 29Si nuclear magnetic resonance (NMR), and theoretical calculations. On the basis of these results, we propose this bond as a member of silicon–silicon bonds such as single bonds, double bonds17, and triple bonds22.We report herein that 1,2,2,3,4,4-hexa-trans-1,3-dibromo-1,2,2,3,4,4-hexa-tert-butylcyclotetrasilane (1) with a large excess of potassium has previously been reported to give the corresponding 1,3-dipotassiocyclotetrasilane23. When the reduction of 1 was carried out with two equivalents of potassium, 1,2,2,3,4,4-hexa-tert-butylbicyclo[1.1.0]tetrasilane (2) was obtained as orange crystals in 50% yield  Å. This value is far larger than the longest silicon–silicon σ bonds (2.697 Å24 and 2.7288(15) Å25) and distances between inverted tetrahedral silicon atoms (2.636(1) Å26 and 2.7076(8) Å27). It is noted that the silicon–silicon and germanium–germanium double bonds in bicyclo[1.1.0]tetrasil-1(3)-ene and a related Ge2Ga2 compound have a π bond and a weak σ bond, and are considerably elongated29. The Si(1)–Si(2) bond length (2.3090(7) Å) is shorter than normal silicon–silicon bonds (2.358 Å)30, probably due to the connection of the two bridgehead silicon atoms. The bridgehead silicon atoms have an almost planar structure: the sum of Si2–Si1–Si2” (103.65(4)°), Si2−Si1−C1 (128.07(2)°), and Si2”–Si1–C1 (128.07(2)°) bond angles is 359.79°. The planar structure of the bridgehead silicon atoms shows that these silicon atoms are nearly sp2-hybridized, which is also discussed later by natural bond orbital (NBO) analysis.The structure of phy Fig. . This mop orbital of the bridgehead silicon atom interact with each other in spite of this unusually long distance? To clarify this point, we measured EPR spectra and magnetic susceptibility. Compound 2 showed no EPR signals in solid state and in a benzene solution  and 66.87% (C) of p-character, indicating sp2 hybrid orbitals. The orbital used for the silicon–silicon π single bond has 0.32% of s-character and 99.46% of p-character. Wiberg bond index of the silicon–silicon π single bond is 0.67. These results show that 2 is a compound with a silicon–silicon π–single bond rather than a singlet diradical.The electronic interaction between the bridgehead silicon atoms was studied by theoretical calculations. The optimized structure of 2 is shown in Fig. 2 shows absorption maxima at 351 and 485 nm. These absorption maxima are assigned to the HOMO to LUMO (485 nm) and HOMO to LUMO + 1 (351 nm) transitions on the basis of a time-dependent density functional theory (TD-DFT) calculation (Supplementary Data i-Pr)2MeSi]2Si = Si[SiMe(i-Pr)2]2 (λmax 412 nm)31, [(t-Bu)Me2Si]2Si = Si[SiMe2(t-Bu)]2 (λmax 424 nm)31, [(i-Pr)3Si]2Si = Si[Si(i-Pr)3]2 (λmax 484 nm)31, and bicyclo[3.3.0]octasil-1(5)-ene (λmax 468 nm)32. Probably, the presence or absence of the silicon–silicon σ bond does not affect the π–π* transition considerably.We obtained other experimental evidence for the silicon–silicon π single bond. The UV/Vis spectrum of 29Si NMR spectrum, 2 shows the signal of the bridgehead silicon atoms at δ 117.4 ppm 16. The signal of 2 at δ 117.4 ppm exists in the intermediate region. This result is explained by substituents on sp2 silicon atoms. Each sp2 silicon atom of tetraalkyl- and tetraaryldisilenes is attached by two carbon atoms and one silicon atom, whereas each sp2 silicon atom of tetrasilyldisilenes is attached by three silicon atoms. In the case of 2, each sp2 silicon atom is attached by two silicon atoms and one carbon atom. Therefore, the intermediate δ value of 2 might be reasonable.In the 2 is interesting from the following viewpoints. Table 2 and related cyclotetrasilanes 1, 3, and 433. These cyclotetrasilanes have been reported to have planar cyclotetrasilane rings and, therefore, the steric repulsion between neighboring tert-butyl groups is large due to the eclipsed conformation. When the van der Waals radius of the substituents on the 1,3-silicon atoms  becomes smaller, the geometry of the 1,3-silicon atoms change from pyramidal to trigonal monopyramidal structures as shown by the Σ (Si) values. This is caused by the reduction of the steric repulsion between neighboring tert-butyl groups. When the hydrogen atoms on the silicon atoms of 3 are removed, the silicon atoms of 2 become nearly planar. Table θ1 value becomes slightly larger and the θ4 value becomes slightly smaller, because of the Thorpe–Ingold effect of the SiX(t-Bu) and Si(t-Bu)2 moieties36; finally, the cyclotetrasilane ring becomes rhombohedral in 237. The increment of the θ1 value from 3 (93.7(1)°) to 2 (103.65(4)°) is significantly large in this series. This structural change corresponds to the formation of the transannular silicon–silicon π single bond.The planar structure of 2 is also interesting from the viewpoint of the bond-stretch isomers of bicyclo[1.1.0]tetrasilane. It was predicted by theoretical calculations that bicyclo[1.1.0]tetrasilane has two bond-stretch isomers, i.e., a short-bond isomer and a long-bond isomer45. The short-bond isomer has a small bridgehead Si–Si bond length (r), a small fold angle between two three-membered rings (ϕ), and a large H−Si−Si bond angle (θ). On the other hand, the long-bond isomer has large r, large ϕ, and small θ values  at various bridgehead silicon–silicon bond lengths. The structures at the inflection point at 2.35 Å and the energy minimum at 2.86 Å correspond to the short- and long-bond isomers , both short-bond and long-bond isomers are highly destabilized. Instead, the planar structure becomes the energy minimum. In the planar structure, the steric repulsion among the bridgehead tert-butyl groups and those at the 2,2,4,4-positions can be avoided. Therefore, the planar isomer may be added to the bond-stretch isomers as a different isomer.We also calculated the energy curves of tert-butyl groups. As this compound was isolated as stable crystals, synthesis of related compounds may be possible. Such studies are now in progress.In conclusion, bicyclo[1.1.0]tetrasilane having a silicon–silicon π single bond was synthesized by introduction of six 1 , potassium , benzene (6 mL), and tetrahydrofuran (THF) (4 mL) was stirred at 50 °C for 2 h in a glovebox. Insoluble materials were removed by filtration through a glass filter. The filtrate was concentrated by slow evaporation of the solvents to give 2  as orange crystals. 2. 1H NMR : δ 1.30 , 1.32 . 13C NMR : δ 24.7, 28.5, 32.3, 33.9. 29Si NMR : δ 13.7, 117.4. UV/Vis (benzene): λmax (ε) 351 (1110 mol−1 L cm−1), 485 nm (1040). High-resolution mass spectrometry: found 454.3307, calcd for C24H54Si4 454.3303.A mixture of 2 were obtained from a benzene–THF (6 : 4) solution by slow evaporation under an argon atmosphere in a glovebox. A crystal specimen was mounted in a loop and was used for data collection on a Rigaku R-AXIS IV++ imaging plate diffractometer using graphite-monochromated Mo Kα radiation. The data were corrected for Lorentz and polarization effects. An empirical absorption correction based on multi-scan was also applied. The structure was solved by a direct method using SHELXS-9749. Non-hydrogen atoms were refined anisotropically by the full-matrix least-squares method on F2 for all reflections using SHELXL-2014/750. All hydrogen atoms were generated by AFIX instructions and were not refined. All calculations were carried out using Yadokari-XG 200951. Crystal data for 2 (123 K): C24H54Si4, fw = 455.03, monoclinic, space group C2/m, a = 16.662(3), b = 11.5749(17), c = 8.5032(15) Å, β = 115.5534(9)°, V = 1479.5(4) Å3, Z = 2, Dcalcd = 1.021 g cm−3, R1 = 0.053 , wR2 = 0.129 , goodness-of-fit = 1.11.Orange crystals of 52 on a Fujitsu PRIMERGY RX300 system of the Research Center for Computational Science, Japan. The structures were optimized at the B3LYP/6-31G(d) level and the optimization was confirmed by frequency calculations. The results are summarized in Supplementary Tables 2 was carried out at the B3LYP/6-31 + G level using the X-ray structure. The result is summarized in Supplementary Data 2 was performed at the B3LYP/6-31G(d) level using the optimized structure. The result is summarized in Supplementary Data All theoretical calculations were performed using Gaussian 09Supplementary InformationPeer Review FileDescription of Additional Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4


Their structures were elucidated by spectroscopic analysis and chemical method. The new compounds represent the first amine alkaloids with a rhamnosyl moiety (1–2) or with a N-oxide motif (2–4) from the genus Ficus. Compound 2 showed potent inhibitory effect in nuclear factor-κB (NF-κB) pathway luciferase assay with IC50 value of 0.52 ± 0.11 μM.Four new alkaloids, ficuhismines A–D (The online version of this article (10.1007/s13659-020-00233-5) contains supplementary material, which is available to authorized users. Ficus (Moraceae) comprises approximately 1000 species all over the world. They mainly distribute in tropical and subtropical areas and show diversity particularly in Southeast Asia + . The IR spectrum of 1 showed absorption bands for OH (3385 cm−1), carbonyl (1675 cm−1), and aromatic (1599 cm−1) functionalities. The 1D NMR , a para-substituted benzene ring , a rhamnopyranosyl moiety , a dimethylamino moiety , and five methylenes. The anomeric configuration of the l-rhamnopyranosyl moiety was determined as α by the chemical shifts of C-3′ and C-5′ [1 was an amine alkaloid rhamnoside. In the HMBC spectrum -1-(4-hydroxyphenyl)-1-hexanone. The H-1′/C-11 HMBC correlation indicated that the sugar was connected to C-11. Acid hydrolysis of 1 afforded the aglycone and sugar units. The sugar was verified as l-rhamnose by TLC analysis and comparing its specific rotation ([α]D25 =  + 6.0) with that of the authentic l-rhamnose ([α]D25 =  + 8.0). Thus, the structure of 1 was elucidated as depicted, and the compound was given the name ficuhismine A.Compound 2 was assigned the molecular formula C20H31NO7 by the (+)-HRESIMS , indicating six indices of hydrogen deficiency. Analysis of its NMR data (Table 2 possessed the same α-rhamnopyranosyl and para-substituted benzene moieties as 1. The only difference was the 6-(dimethylamino)-1-hexanone side chain. In the 13C NMR spectrum of 2, the obviously deshielded carbon signals of the dimethylamino moiety (ΔδC + 13.0) and the methylene at C-2 (ΔδC + 11.6) were observed. In consideration the indices of hydrogen deficiency and an additional oxygen atom in the molecular formula of 2, the presence of a N-oxide motif was inferred. The HMBC data shown in Fig. 2. The sugar was identified as l-rhamnose by the same protocol as that of 1. Compound 2 was thus elucidated as an N-oxide of 2 and named ficuhismine B.Compound 3 was assigned the molecular formula C14H21NO3 by the (+)-HRESIMS ion at m/z 252.1595 [M+H]+ . Its 1D -HRESIMS ion at m/z 212.1281 [M+H]+ . The 1H and 13C NMR spectra of 4  D25 = − 61.0 ; UV (MeOH) λmax (log ε) 206 (4.28), 262 (4.32) nm; IR (KBr) νmax 3385, 2915, 1675, 1599, 1383, 1181, 1141, 1075, 632 cm−1; 1H NMR and 13C NMR data: Table m/z 382.2232 [M+H]+ .White solid; [α]D25 = − 70.0 ; UV (MeOH) λmax (log ε) 206 (4.32), 264 (4.41) nm; IR (KBr) νmax 3372, 2935, 1675, 1600, 1421, 1196, 1179, 1140, 1073, 1014, 971, 835 cm−1; 1H NMR and 13C NMR data: Table m/z 398.2165 [M+H]+ .White solid; + .White solid; UV (MeOH) λmax (log ε) 204 (3.81), 216 (3.80), 274 (3.97) nm; IR (KBr) νmax 3409, 2950, 2937, 2855, 2581, 1599, 1524, 1454, 1387, 1280, 1033 cm−1; 1H NMR and 13C NMR data: Table m/z 212.1281 [M+H]+ .White solid; UV (MeOH) ) 204 3.8, 216 (3.1 (1 mg) was added to a solution of H2O (3 mL), dioxane (3 mL), and HCl (0.5 mL), and then kept at 60 °C for 4 h. The reaction mixture was quenched with NaHCO3 and extracted with EtOAc. The aqueous phase was concentrated under reduced pressure. The residue was dissolved in H2O for TLC analysis with the authentic l-rhamnose  and for optical rotation measurement {the aqueous phase: [α]D25 =  + 6.0 (c 0.1 H2O); l-rhamnose: [α]D25 =  + 8.0 (c 0.1 H2O)}. Compound 2 was operated in the same way as 1.Compound κB cells were prepared as reported [50 values were calculated using Graphpad prism 8.HEK293/NF-reported . The lucreported . The IC5Electronic supplementary material 1 (DOCX 6474 kb)Below is the link to the electronic supplementary material.


Mice were infected with HSV-2 and sacrificed when showing signs of neuroinflammation. Cells and/or tissue from spinal cord, spleen, and blood were analyzed for expression of activation markers, chemokine receptors, and chemokines. High numbers of CD8+ T cells were present in the spinal cord following genital HSV-2-infection. CD8+ T cells were highly activated and HSV-2 glycoprotein B -specific effector cells, some of which showed signs of recent degranulation. They also expressed high levels of many chemokine receptors, in particular CCR2, CCR4, CCR5, and CXCR3. Investigating corresponding receptor ligands in spinal cord tissue revealed markedly increased expression of the cognate ligands CCL2, CCL5, CCL8, CCL12, and CXCL10. This study shows that during herpesvirus neuroinflammation anti-viral CD8+ T cells accumulate in the CNS. CD8+ T cells in the CNS also express chemotactic receptors cognate to the chemotactic gradients in the spinal cord. This indicates that anti-viral CD8+ T cells may migrate to infected areas in the spinal cord during herpesvirus neuroinflammation in response to chemotactic gradients.Herpes simplex virus type 2 (HSV-2) is a neurotropic virus that can cause meningitis, an inflammation of the meninges in the central nervous system. T cells are key players in viral clearance, and these cells migrate from peripheral blood into the central nervous system upon infection. Several factors contribute to T cell migration, including the expression of chemokines in the inflamed tissue that attract T cells through their expression of chemokine receptors. Here we investigated CD8 Herpes simplex virus type 2 (HSV-2) is a double-stranded DNA virus that infects through mucosal tissue to establish a latent infection in the central nervous system (CNS) . This study was approved by the ethical committee for animal experiments .About 12–20-week-old female C57bl/6 mice (Charles River) were infected with HSV-2 strain 333 as previously described  diluted in 0.1% saponin (Sigma-Aldrich) in PBS for 1.5 h at room temperature. When using mouse monoclonal antibody, Mouse on Mouse (M.O.M.®) blocking reagent kit  was applied for pre-incubation. Sections were then incubated overnight at 4 °C with primary antibodies diluted 1:200 in working solution . Antibodies used included rabbit polyclonal anti-HSV-1/2 , mouse monoclonal anti-GFAP (clone 2A5) , and rat anti-CD8-biotin (clone 53-6.7) . Biotinylated Abs were detected with Alexa Fluor™ 555 Tyramide SuperBoost™ Kit (ThermoFisher Scientific), while other primary antibodies were detected using Alexa Fluor 647 anti-mouse (cat. # A-21235) or Alexa Fluor 488 anti-rabbit (cat. # A31572) polyclonal antibodies (ThermoFisher Scientific). After final washing in PBS, slides were closed in SlowFade™ Diamond Antifade Mountant with 4-6-diamidino-2-phenylindole . Sections were imaged by confocal microscopy using a Zeiss Laser Scanning Inverted Microscope LSM-700 equipped with 40X/1.3 Oil NA objective and Black Zen software (Carl Zeiss) at the Center for Cellular Imaging, University of Gothenburg.6 million cells/sample were subsequently used for flow cytometric staining. Spinal cords were resuspended in 2 ml PBS, and 200 µL were used for staining one sample. After blocking of unspecific binding for 10 min using 5 µg/mL rat anti-mouse CD16/CD32 (cat no 553142), cells were stained for 20 min using the following mAbs, CD44 PE (cat no 561860), CD8 BV421 (cat no 563898), CCR7 PerCp-Cy5.5 (cat no 560812) (BD Biosciences), CCR2 PE (cat no FAB5538P), CXCR3 PE (cat no FAB1685P), CXCR4 FITC (cat no FAB21651F) (R&D Systems), CCR4 APC (cat no 131211), CCR4 PE (cat no 131203), CCR5 APC (cat no 107011), CCR6 PerCp-Cy5.5 (cat no 129810), CXCR3 FITC (cat no 126535), CXCR4 APC (cat no 146507), CD107a PerCP-Cy5.5 (cat no 121625), CD3 FITC (cat no 100306), (Biolegend), CD62L APC , and SSIEFARL APC (ProImmune). For intracellular staining, BD cytofix/cytoperm fixation/permeabilization kit (BD) was used according to manufacturer’s instructions. Intracellular staining was done using FITC rat anti-granzyme B  and PE rabbit anti-active caspase-3 (cat no 561011 BD). Cell viability was assessed by staining with Fixable Viability Dye eFluor 506  , and 10N, SysmexWhole blood was collected and processed into serum by allowing clotting in room temperature, followed by separation of the serum phase by centrifugation. Extractions of tissue was achieved by collecting spinal cords in PBS with 0.1 mg/ml soybean trypsin inhibitor (Sigma), 1.5 mM Pefabloc (Coatech AB), 50 mM EDTA, and 0.1% bovine serum albumin. Samples were weighed and frozen. Upon thawing, samples were permeabilized using 2% saponin over night at 4 °C, and the supernatant was subsequently collected by centrifugation. Chemokine and cytokine analysis was performed using a mouse magnetic Luminex assay (CCL2 (lowest level of quantification (LLOQ) = 415 pg/ml), CCL3 (LLOQ = 11 pg/ml), CCL4 (LLOQ = 288 pg/ml), CCL7 (LLOQ = 8 pg/ml), CCL8 (LLOQ = 8 pg/ml), CCL11 (LLOQ = 19 pg/ml), CCL12 (LLOQ = 8 pg/ml), CCL20 (LLOQ = 216 pg/ml), CCL22 (LLOQ = 29 pg/ml), CXCL1 (LLOQ = 72 pg/ml), CXCL2 (LLOQ = 6 pg/ml), CXCL5 (LLOQ = 63 pg/ml), CXCL10 (LLOQ = 153 pg/ml), CXCL12 (LLOQ = 525 pg/ml), GM-CSF (LLOQ = 21 pg/ml), IFN-gamma (LLOQ = 20 pg/ml), IL-1alpha (LLOQ = 63 pg/ml), IL-1beta (LLOQ = 302 pg/ml), IL-2 (LLOQ = 11 pg/ml), IL-4 (LLOQ = 87 pg/ml), IL-6 (LLOQ = 57 pg/ml), IL-10 (LLOQ = 35 pg/ml), TNF-alpha (LLOQ = 3 pg/ml)) from R&D Systems according to manufacturer’s instructions and analyzed using Bio-Plex 200 system (Bio-Rad). The levels of CCL5 were assessed using a DuoSet ELISA kit  according to manufacturer’s instruction.T test was used when two groups were compared, and one-way ANOVA with Dunnett’s or Tukey´s multiple comparisons test was used when three or more groups were compared. Statistical analysis was performed using GraphPad Prism version 7 (GraphPad Software).All data are expressed as mean ± SEM. Unpaired + T cell profile in the CNS, mice were infected with HSV-2. Neurological symptoms were observed 7–9 days post infection . Animals were then euthanized, and cells from spinal cord (CNS tissue) and spleen  were used for flow cytometric analysis both before and after infection. Low numbers of CD8+ T cells were detected in spinal cord from healthy animals. Following HSV-2 infection the numbers increased almost 100-fold, compared with healthy animals , apoptotic (non-viable cells expressing caspase-3), or dead  and CXCL10 (which is a CXCR3 ligand) in the spinal cord of HSV-2-infected mice  effector/memory cells, similar to what has been shown in other inflammatory diseases of the CNS Below is the link to the electronic supplementary material.


Structures are reported for two sets of substituted 4-styrylquinolines formed by reactions of (2-aminophenyl)chalcones either with pentane-2,4-dione or, regiospecifically, with ethyl 3-oxobutanoate. E)-3-Acetyl-4-[2-(4-methoxyphenyl)ethenyl]-2-methylquinoline, C21H19NO2, (I), (E)-3-acetyl-4-[2-(4-bromophenyl)ethenyl]-2-methylquinoline, C20H16BrNO, (II), and (E)-3-acetyl-2-methyl-4-{2-[4-(trifluoromethyl)phenyl]ethenyl}quinoline, C21H16F3NO, (III), are isomorphous and in each structure the molecules are linked by a single C—H⋯O hydrogen bond to form C(6) chains. In (I), but not in (II) or (III), this is augmented by a C—H⋯π(arene) hydrogen bond to form a chain of rings; hence, (I)–(III) are not strictly isostructural. By contrast with (I)–(III), no two of ethyl (E)-4-[2-(4-methoxyphenyl)ethenyl]-2-methylquinoline-3-carboxylate, C22H21NO3, (IV), ethyl (E)-4-[2-(4-bromophenyl)ethenyl]-2-methylquinoline-3-carboxylate, C21H18BrNO2, (V), and ethyl (E)-2-methyl-4-{2-[4-(trifluoromethyl)phenyl]ethenyl}quinoline-3-carboxylate, C22H18F3NO2, (VI), are isomorphous. The molecules of (IV) are linked by a single C—H⋯O hydrogen bond to form C(13) chains, but cyclic centrosymmetric dimers are formed in both (V) and (VI). The dimer in (V) contains a C—H⋯π(pyridyl) hydrogen bond, while that in (VI) contains two independent C—H⋯O hydrogen bonds. Comparisons are made with some related structures, and both the regiochemistry and the mechanism of the heterocyclic ring formation are discussed.Structures are reported for two matched sets of substituted 4-styrylquinolines which were prepared by the formation of the heterocyclic ring in cyclocondensation reactions between 1-(2-aminophenyl)-3-arylprop-2-en-1-ones with 1,3-dicarbonyl compounds. ( Compounds such as (I)–(III), containing an acetyl group, can act as useful synthetic intermediates, as they can undergo condensation with a further substituted aldehyde to form a chalcone substituent at the 3-position, as exemplified by compound (VIII)  Figs. 4–6 ▸ ▸, et al., 2020Samples of compounds (I)–(VI) were prepared and crystallized following a recently published procedure (Meléndez 3) or 0.99 Å (CH2), and with Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups, which were permitted to rotate but not to tilt, and 1.2 for all other H atoms. For compounds (VII) and (VIII), the published structures  using PLATON –(VI) are summarized in Table 1A)  with an unsymmetrical diketo compound, such as ethyl 3-oxobutanoate, can, in principle, give two types of product: reaction of the amino group at the acetyl carbonyl group leads to ethyl esters, as exemplified by compounds (IV)–(VI), but reaction of the amino group at the ester carbonyl group would lead to elimination of ethanol with the formation of a 2-quinolone of type (X) et al., 2009et al., 2012i.e. (IV)–(VI), where X = OEt, although compounds (IV)Compounds (I)–(III), where  Scheme 1–3 ▸ ▸, None of the molecules of (I)–(VIII) exhibits any internal symmetry, so that they are all conformationally chiral; in each case, the reference molecule was selected as one having a positive sign for the C3—C4—C41—C42 torsion angle Table 2, althougC(6) The supramolecular assembly of compounds (I)–(VI) is determined by C—H⋯O and C—H⋯π hydrogen bonds Table 3. In eachgs Fig. 7. HoweverC(13) chain running parallel to the [100] direction on Fig. 8. This ste Table 3, while ae Table 3.In the structure of compound (VI)g Fig. 10.Introduction), have been reported on a simple proof of constitution basis, without discussion  hydrogen bond (Table 3a-glide plane at z = C(6)C(9)[b-glide plane at x = z < z < 1.0, but there are no direction-specific interactions between adjacent sheets. Even the shortest intermolecular contacts  and (VIII), which, as noted above  and (VIII)  global, I, II, III, IV, V, VI. DOI: 10.1107/S2053229620010803/sk3755Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2053229620010803/sk3755IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2053229620010803/sk3755IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2053229620010803/sk3755IVsup5.hklStructure factors: contains datablock(s) IV. DOI: 10.1107/S2053229620010803/sk3755Vsup6.hklStructure factors: contains datablock(s) V. DOI: 10.1107/S2053229620010803/sk3755VIsup7.hklStructure factors: contains datablock(s) VI. DOI: Click here for additional data file.10.1107/S2053229620010803/sk3755Isup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229620010803/sk3755IIsup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229620010803/sk3755IIIsup10.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229620010803/sk3755IVsup11.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229620010803/sk3755Vsup12.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229620010803/sk3755VIsup13.cmlSupporting information file. DOI: 10.1107/S2053229620010803/sk3755sup14.cifCrystal structure: contains datablock(s) VII, VIII. DOI: 2021528, 2021527, 2021526, 2021525, 2021524, 2021523CCDC references:


The u-Cre was measured enzymatically using creatininase, creatinase, sarcosine oxidase, and peroxidase. None of the participants was diagnosed with any kidney disease. Median u-γGT and u-γGT/u-Cre values  were 29.7 (5.3–144.0) U/L and 57.9 (32.9–122.7) U/g creatinine, respectively. The distribution of u-γGT tended to decline with age. There was a statistically significant difference in the u-γGT value between the 40-59- and 60-74-year-old groups. In contrast, there was no significant difference in the u-γGT/u-Cre between each age group. The u-Cre level also declined with age. It is suggested that the decline of u-γGT with aging would be masked by the u-Cre correction.Urinary gamma-glutamyltransferase (u-γGT) concentration (U/L) and excretion  are useful markers for kidney disease. However, there is limited information available on u-γGT and u-γGT/u-Cre distribution in the elderly Japanese population. In this study, we investigated the distribution of u-γGT and u-γGT/u-Cre in 113 Japanese women aged 40–74 years. The u-γGT was assessed from spot urine samples (collected from 09:00 to 14:00) spectrophotometrically according to the Japan Society of Clinical Chemistry reference measurement procedure using  •Median  u-γGT value was 29.7 (5.3–144.0) U/L.•Median  u-γGT/u-Cre ratio was 57.9 (32.9–122.7) U/g creatinine.•Distribution of u-γGT tends to decline with age.•u-Cre levels also declined with age.•Decline of u-γGT with aging would be masked by the u-Cre correction. In thFor practical applications as a biomarker, it is crucial to determine the u-γGT and u-γGT/u-Cre distribution in individuals without renal disease as a reference; however, this information is currently limited. In particular, there is limited information available on the u-γGT and u-γGT/u-Cre distribution in old Japanese adults. Given that u-γGT undergoes a physiological change during aging  and the In Japan, “specific health checkups and specific health guidance” are carried out for all (40–74)-year-old insured individuals and their dependents to improve screening and detection of metabolic syndrome. Therefore, in this study, we used the data available from these “specific health checkups” to determine the distribution of γGT and u-γGT/u-Cre in (40–74)-year-old Japanese women. We then compared the u-γGT concentration and excretion levels in each age group within this cohort.22.1Urine samples were obtained from (40–74)-year-old women (n = 152) who received specific health checkups in Choshi, Japan in July and August 2017. Only women were included in the study since the primary participants of the health checkups offered by the local government carries are housewives and elderly individuals.2 and/or urinary Alb ≥30 mg/g creatinine, which are two diagnostic criteria of chronic kidney disease in Japan  were o2.3l-γ-glutamyl-3-carboxy-4-nitroanilide (GluCANA) as the substrate [app.ε) of 5-amino-2-nitrobenzoate (5-ANB), used as the enzyme reaction indicator of our spectrophotometer, was 7525 L·mol-1·cm-1 (37 °C). The γGT activity was then calculated using the following formula:app. ε is the apparent molar absorption coefficient (L·mol-1·cm-1), V is the total volume of the reaction solution (mL), and v is the sample volume (mL). In this analytical method, the lower and upper limits of quantitation were 2.3 and 800 U/L, respectively. The inter- and intra-assay coefficient of variations (CVs) were 2.15% and 1.71%, respectively.The u-γGT was measured according to the Japan Society of Clinical Chemistry (JSCC) reference measurement procedure using ubstrate . In brieUrinary creatinine (u-Cre) levels were also measured for the creatinine correction of the u-γGT by an enzymatic method using creatininase, creatinase, sarcosine oxidase, and peroxidase  and the clinical autoanalyzer, HITACHI 3100 (Hitachi High-Technologies Corp.). The urine sample was diluted 10-fold with ultrapure water. Controls WAKO-I and –II  were used for γGT and creatinine measurement quality control. Urinary albumin (u-Alb) levels were also measured by the immune nephelometric method using a polyclonal anti-human albumin antibody  and the clinical autoanalyzer, HITACHI 3100. Control for Auto WAKO microalbumin  was used for u-Alb measurement quality control.2.4U test. Differences with a P-value (P) < 0.05 were considered statistically significant. SPSS  was used for statistical analyses.The normality of each clinical test value was checked by histogram and tested using the Shapiro-Wilk test. Parametric data were indicated as the mean ± standard deviation, and nonparametric data were indicated as median and interquartile range. Correlations between u-γGT, u-Cre, u-γGT/u-Cre, and each medical examination variable were determined by Spearman correlation coefficients using subject means. Statistical significance was determined by a two-sided Mann-Whitney 33.1The fundamental statistics of physical exams, and blood and urinary biochemical parameter levels for the 113 participants are shown in U test (P = 0.007). In contrast, the u-γGT/u-Cre showed a slight decrease between the 60s and 70s, whereas the difference in u-γGT/u-Cre values between (40–69)- and (70–74)-years-old groups was not significant according to the Mann-Whitney U test (P = 0.295).The distributions of u-γGT and u-γGT/u-Cre tended to decline with age. The u-γGT drastically decreased between the 50s and 60s. There was a statistically significant difference in the u-γGT value between the (40–59)- and (60–74)-years-old groups according to the Mann-Whitney 3.2P = 0.012), height , weight , u-Cre , u-Alb , and u-γGT/u-Cre . No significant correlations were observed between u-γGT and abdominal girth, systolic and diastolic blood pressures, HDL-C, LDL-C, TG, AST, ALT, s-γGT, HbA1c, s-Cre, eGFR, or u-Alb/u-Cre. No significant correlation was observed between u-γGT/u-Cre and medical examination parameters.A statistically significant correlation was observed between u-γGT and age  was observed between u-γGT and u-Cre suggesting creatinine correction is reasonable for u-γGT. Conti et al. reported that correcting any tubular marker to u-Cre is not appropriate; however, u-Cre correction is appropriate for pure glomerulopathy [P = 0.012), and a statistically significant difference in the u-γGT value between the (40–59)- and (60–74)-year-old groups (P = 0.007). The decline of u-γGT with age has previously been described [P = 0.012). The negative correlation between u-Cre and age is considered to reflect a decrease in the quantity of muscle with aging [In this study, we measured the u-γGT levels in (40–74)-year-old Japanese females n = 113). We also measured the creatinine-corrected value u-γGT/u-Cre, which is commonly used in clinics since the u-γGT of spot urine can be affected by the dilution and concentration of urine. A statistically significant positive correlation , a statistically significant correlation was observed between u-γGT and height or weight. Indeed, the number of nephrons has been reported to be proportional to body habitus ,26, whicThe u-γGT correlated with u-γGT/u-Cre and u-Alb. The correlation between u-γGT and u-γGT/u-Cre is quite natural because u-γGT/u-Cre was calculated from u-γGT. The correlation of u-γGT with u-Alb has also been reported in the younger population  and is kU test. Second, there were only weak correlations between age and u-γGT or u-Cre. These observations might cast doubt on the suggestion of this study that creatinine-corrected u-γGT values are overestimated in elderly women.This study has some limitations. First, the number of subjects (n = 30) in the 40–59-year-old age group was small and therefore, may not be good representative of the population from which they were drawn. The difference in the sample size between the (40–59)-year-old group (n = 30) and the 60-74-year-old group (n = 83) may reduce the statistical power of the Mann-Whitney In this study, we determined the urinary γGT distribution in (40–74)-year-old Japanese women receiving specific health checkups, whereby we aimed to provide basic data for the establishment of urinary γGT reference ranges for each included decade of age. Median u-γGT tended to decline with age, similar to previous studies ,19. For The study plan was approved by the Ethical Committee for Medical and Health Research Involving Human Subjects of Chiba Institute of Science  and Department of Sports Science at Juntendo University . All the participants provided informed consent for the use of their urine samples and medical examination information. The Health and Welfare Center of Choshi-city  implemented the organization of the medical examinations and provided permission for the use of the data in this study.Akihisa Hata: Investigation, Formal analysis, Data curation, Writing - original draft, Visualization. Maki Miyauchi: Investigation, Resources. Yoshio Suzuki: Resources, Investigation, Project administration. Yuki Otomo: Investigation, Validation. Noboru Fujitani: Conceptualization, Writing - review & editing, Supervision.


Spinal cord injury (SCI) is a severe central nervous system trauma. The present study aimed to evaluate the effect of HIF-1α on inflammation in spinal cord injury (SCI) to uncover the molecular mechanisms of anti-inflammation.HIF-1α was reduced in SCI model rats and HIF-1α activation reduced TNF-α, IL-1β, IL-6 and IL-18 levels in SCI model rats. Meanwhile, Circ 0001723 expression was down-regulated and miR-380-3p expression was up-regulated in SCI model rats. In vitro model, down-regulation of Circ 0001723 promoted TNF-α, IL-1β, IL-6 and IL-18 levels, compared with control negative group. However, over-expression of Circ 0001723 reduced TNF-α, IL-1β, IL-6 and IL-18 levels in vitro model. Down-regulation of Circ 0001723 suppressed HIF-1α protein expressions and induced NLRP3 and Caspase-1 protein expressions in vitro model by up-regulation of miR-380-3p. Next, inactivation of HIF-1α reduced the pro-inflammation effects of Circ 0001723 in vitro model. Then, si-NLRP3 also inhibited the pro-inflammation effects of Circ 0001723 in vitro model via promotion of autophagy.We concluded that HIF-1α reduced inflammation in spinal cord injury via miR-380-3p/ NLRP3 by Circ 0001723. Spinal cord injury (SCI) can be divided into primary injury and secondary injury according to its pathogenesis . Of themCircular RNAs (circRNAs), one kind of non-coding RNA, have been reported as numerous biological processes, such as inflammation, proliferation, apoptosis, and cell cycle regulation . miRNA iMale Sprague–Dawley rats  were purchased from Animal Experimental Center of Zhejiang University. All rats were housed at 22–23 °C, 55–60% humidness, 8:00–20:00 light/ dark cycle, and received standard rodent chow and access to water ad libitum. This study was approved by Ethical Committee of The First Affiliated Hospital of Zhejiang University.Rats were fixed in the prone position and anesthetized by 35 mg/kg of pentobarbital. Skin was cut to 2–3 cm middle incision in the back, and vertebral T7–T9 were exposed. After stabilizing vertebral T7 and T9, a laminectomy was performed at the thoracic level T8. A syringe needle was used to induce the injury, which was released from a height of 12.5 mm above the surface of the cord, inflicting a moderate contusion. Hemostatic suture was performed layer by layer, and alcohol was then applied for disinfection.After 24 h of induction SCI, BBB test was performed as locomotor rating scale of 0 (no observable hind-limb movements) to 21  . After 2Next, part tissue fixed with 4% paraformaldehyde for 24 h, dehydrated, and embedded in OCT compound. Tissues were cut in the sagittal or axial plane into 10-μm sections. Thick sections were stained with hematoxylineosin (H&E) for 15 min. Tissue sample sections were examined by a light microscope .Total RNA was amplified into Affymetrix HG-U133 Plus 2.0 GeneChip arrays . Data were analyzed through Database for Annotation, Visualization, and Integrated Discovery (DAVID Database), and QIAGEN’s Ingenuity Pathway Analysis .Real-time PCR analysis. Total RNA extracted from tissue samples and cell samples with Trizol (Invitrogen USA) and converted into cDNA by using M-MLV reverse transcriptase and cDNA Synthesis Kit (Invitrogen USA). Real-time PCR was conducted by using iCycler RealTime PCR Detection System  with a SYBR ExScript RT-PCR kit . The thermal cycling conditions were as follows: 95.0 °C for 10 min, 40 cycles of 95.0 °C for 15 s, 60.0 °C for 30 s and 72.0 °C for 5 min. The relative expression level was presented as 2−ΔΔCt [s 2−ΔΔCt .ELISA kits. Tissue samples and cell samples were collected, and proteins were extracted by RIPA lysis buffer. 10 μg protein was used to analyze TNF-α, IL-1β, IL-6 and IL-18 levels using ELISA KITS.2 at 37 °C. HIF-1α plasmid, miR-143, anti- miR-143 and negative mimics were transfected into cell using Lipofectamine 3000 . After 48 h of transfection, Neuro-2a cell was induced with 200 ng/mL of LPS for 4 h. Next, after 4 h of transfection, Neuro-2a cell was treated with LC3 inhibitor  or autophagy agonist (1 μM resveratrol) for 4 h, and Neuro-2a cell was induced with 100 ng/mL of LPS for 4 h.PC12 cell was incubated using the Dulbecco’s Modifed Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) at an incubator with 5% COCells were harvested and proteins were extracted by RIPA lysis buffer. Proteins were separated on 10% polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were then blocked with 5% non-fat milk in Tris-buffered saline containing Tween-20 (TBST). at room temperature for 1 h and incubated with anti-HIF-1α, anti-p65, anti-LC3, anti-p62, anti-GAPDH  at 4 °C overnight. Membranes were subsequently incubated with goat anti-rabbit peroxidase-conjugated secondary antibodies  at 37 °C for 1 h. Membranes was detected using an enhanced chemiluminescence kit and Image Lab 3.0 .Cell was washed with PBS and fixed with 4% paraformaldehyde for 15 min. Cell was washed with PBS and blocked with 5%-BSA supplemented with 0.25%-Tris-X100 in PBS for 1 h at room temperature. Cell was washed with PBS and incubated with anti-HIF-1α and anti-LC3 at 4 °C overnight. Cell was washed with PBS and incubated with 555- or 488- goat anti-rabbit peroxidase-conjugated secondary antibodies (1:100) room temperature for 1 h. Cell was washed with PBS and incubated with DAPI assay for 15 min at darkness.All data are represented as mean ± SD for three independent experiments. Student’s t-tests or one-way analysis of variance (ANOVA) and Tukey’s post test were used for all comparisons involving continuous dependent variables. Results were considered statistically significant when the p-value was < 0.05.P < 0.05)  .To evaluate the changes of HIF-1α expression in SCI, we analyzed HIF-1α expression using QPCR. Firstly, by HE staining, spinal cord death was observed in SCI model group   Fig. a–g. HIF-05) Fig. h, i.Fig.P < 0.05) . Down-regulation of Circ 0001723 significantly increased TNF-α, IL-1β, IL-6 and IL-18 levels in vitro model of SCI compared with negative control group (P < 0.05)           Fig. a. There 05) Fig. b. Circ 005) Fig. c, d. Cir05) Fig. e, f. The Fig. a. 05) Fig. a–d. Over05) Fig. e.Fig. 4CP < 0.05)     Fig. a–d. The 05) Fig. e–h.Fig. expression in vitro model of SCI by Circ 0001723 down-regulation compared with Circ 0001723 down-regulation group . The activation of LC3 significantly reduced TNF-α, IL-1β, IL-6 and IL-18 levels in vitro model of SCI by Circ 0001723 down-regulation compared with Circ 0001723 down-regulation group (P < 0.05)  Fig. d–g.Fig. SCI is one of the common severe diseases in Spine Surgery. It is associated with extremely high mortality and disability. Typically, its treatment remains a worldwide challenge. Autophagy is an important defense and protection mechanism of the body . The bodInflammatory chemokine and cytokine can supplement and activate the immune cells and central nervous cells . They haBioinformatics analysis suggests that, the potential target genes of the changed miRNA after SCI include those encoding the components during inflammation, oxidative stress and apoptosis . These pResearch indicates that, HIF-1α expression is increased under hypoxia and ischemia/reperfusion status . InterveWe showed for the first time in our study that Circ 0001723 promoted inflammation, and induced NF-κB protein expression in SCI via suppression of Autophagy by HIF-1α Fig. . These r