In the crystal, the benzamide molecules are linked by N—H⋯O hydrogen bonds to generate tetramers with an approximate square-prismatic shape, which appears to correlate with the tetragonal crystal symmetry. 20H18N6O5·0.5C3H7NO, crystallized as a dimethylformamide (DMF) solvate. In the main molecule, the dihedral angle between the pyridodipyrimidine fused-ring system and the benzamide substituent is 82.26 (11)°. In the crystal, the benzamide molecules are linked by N—H⋯O hydrogen bonds to generate tetramers with an approximate square-prismatic shape, which appears to correlate with the tetragonal crystal symmetry. The DMF molecule is disordered about a crystallographic twofold axis and accepts a C—H⋯O interaction from the benzamide molecule.The title compound, C In particular, the representatives of this family show antiviral benzamide molecule, related by twofold axes, via a non-classical C17–H17⋯O51 hydrogen bond .Each disordered DMF solvent molecule is connected to an adjacent 2--tetrone -tetrone  5-(4-fluorophenyl)-1,3,7,9-tetramethylpyrido[2,3-M) and 1,2-benzenedicarbonyl chloride 2.0 g (10 mM) dissolved in 10 ml DMFA was stirred under reflux for 2 h. The mixture was concentrated under reduced pressure, then 20 ml of iced water was added to it and filtered. The synthesized compound was dissolved in ethanol and crystallized by slow evaporation at room temperature .A mixture of 6-amino-1,3-dimethyl-1,2,3,4-tetrahydro-2,4-pyrimidinedione 1.55 g (10 mUiso(H) = 1.2Ueq(C), and for the methyl groups, C—H = 0.96 Å, Uiso(H) = 1.5Ueq(C). Only the coordinates of the H atoms of the amide group, involved in hydrogen bonding, were determined from difference Fourier syntheses and refined freely.Crystal data, data collection details and structure refinement details are summarized in Table 210.1107/S1600536814020200/hb7283sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S1600536814020200/hb7283Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814020200/hb7283Isup3.cmlSupporting information file. DOI: 1023362CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


By contrast, (Arg-Benz)4-CONH2 and 3--CONH2 prevented spontaneous and seeded Aβ42 and Aβ43 fibrillization. Importantly, 3--CONH2 inhibited formation of toxic Aβ42 and Aβ43 oligomers and proteotoxicity. None of these foldamers inhibited Sup35 prionogenesis, but Sal-3-CONH2 delayed aggregation of fused in sarcoma (FUS), an RNA-binding protein with a prion-like domain connected with amyotrophic lateral sclerosis and frontotemporal dementia. We establish that inhibitors of Aβ42 fibrillization do not necessarily inhibit Aβ43 fibrillization. Moreover, 3--CONH2 inhibits formation of toxic Aβ conformers and seeding activity, properties that could have therapeutic utility.Amyloid fibrils are self-propagating entities that spread pathology in several devastating disorders including Alzheimer's disease (AD). In AD, amyloid-β (Aβ) peptides form extracellular plaques that contribute to cognitive decline. One potential therapeutic strategy is to develop inhibitors that prevent Aβ misfolding into proteotoxic conformers. Here, we design specific aromatic foldamers, synthetic polymers with an aromatic salicylamide  or 3-amino benzoic acid (Benz) backbone, short length (four repetitive units), basic arginine (Arg), lysine (Lys) or citrulline (Cit) side chains, and various N- and C-terminal groups that prevent spontaneous and seeded Aβ fibrillization. Ac-Sal- We report specific aromatic foldamers that inhibit fibrillization by amyloid-β (Aβ) peptides connected with Alzheimer's disease. One foldamer inhibits formation of toxic Aβ-species as well as the self-templating activity of Aβ fibrils, properties that could have therapeutic utility. Protein misfolding can be fatal ,2. ProteHere, we focus on amyloid-β (Aβ) peptides, Aβ42 and Aβ43, which form amyloid fibrils and accumulate in extracellular plaques that are a hallmark of AD –16. AD iAlthough Aβ peptides longer than Aβ42 are found in AD, they are not a major species and their pathogenic role has been ignored. Recently, this view has changed. Aβ43 is a potent contributor to neurotoxicity in AD –15. Aβ43Aβ monomers form amyloid via nucleated conformational conversion . First, Numerous potential inhibitors of Aβ misfolding have been explored, including small molecules, peptides, molecular chaperones, protein disaggregases and antibodies ,6,39–45.2 and 1 mM DTT) to 10 μM. This solution was incubated at 37°C for 3–5 days with agitation (700 r.p.m.) in an Eppendorf Thermomixer. For seeding experiments, preformed fibrils were briefly sonicated or vortex-mixed prior to use. We also prepared Aβ42 or Aβ43 using a protocol that avoids DMSO. Thus, Aβ42 or Aβ43 was dissolved in HFIP followed by evaporation of the solvent to dryness  and dried with MeOH. This product was split in half. The first half was re-swelled in DMF and acetylated by incubating the resin in 5% acetic anhydride in 2.5% DIEA and 92.5% DMF for 10 min. This acetylated portion was rinsed and dried (as above). Next, both halves  were cleaved from the resin using a cocktail of 2:2:2:94 H2O/TIS (tri-isopropyl silane)/anisole/TFA  for 2 h at room temperature. The peptide solution was filtered from the resin and precipitated using 1:1 cold ethyl ether:hexane. The precipitate was dried by lyophilization. The mass and purity of each product was verified by MALDI–TOF MS (Brucker microflex LRF) and analytical HPLC (C18 column). Dried crude foldamer was purified by preparative reverse-phase HPLC, dried by lyophilization and mass and purity was verified as above. All samples were prepared by directly dissolving lyophilized foldamer into TBS buffer to 2 mM.Foldamers For spontaneous fibrillization, soluble Aβ42 or Aβ43 (1 mM) in DMSO was diluted to 5 μM in KHMD containing 25 μM thioflavin-T (ThT) plus or minus foldamer (0–20 μM). For seeded fibrillization, preformed Aβ42 or Aβ43 fibrils (10 μM monomer) were added at a final concentration of 0.1 μM (monomer). Alternatively, Aβ42 or Aβ43 were prepared using just HFIP and were assembled at 5 μM in PBE containing 25 μM ThT plus or minus foldamer (20 μM). NM was purified as described . NM (5 μGST–TEV–FUS was purified as described . AggregaReactions were adhered on to 300-mesh-formvar carbon-coated EM grids overnight before being negatively stained with 2% uranyl acetate for 2 min and rinsed with milli-Q distilled water. Micrographs were acquired using a JEOL 1010 TEM (Jeol USA).The oligomer-specific A11 antibody was used to detect toxic Aβ42 or Aβ43 oligomers by ELISA as described . Foldame2 at 37°C. Cells were differentiated in serum-free DMEM with N2 supplement and 10 μM all-trans-retinoic acid before use. Cells were plated at (10000 cells/well) in 96-well plates and grown overnight. Medium was removed and Aβ conformers or controls were added and cells were incubated for 24 h at 37°C. Toxicity was assessed using an MTT kit  or via lactate dehydrogenase (LDH) release using the CytoTox-ONE™ kit (Promega). Toxicity values were normalized to the buffer control without Aβ.SH-SY5Y human neuroblastoma cells were maintained in Dulbecco's modified Eagle's medium (DMEM) plus 10 mM Hepes, 10% FBS, 4 mM glutamine, penicillin (200 units/ml) and streptomycin (200 μg/ml) in 5% CO2 or COMe [Ac]) and C- terminal groups. We selected this design for four reasons. First, the aromatic backbone is similar to ones employed by Nowick et al. [As potential inhibitors of Aβ42 and Aβ43 amyloidogenesis, we explored aromatic foldamers  and 2. Sk et al. –53 in prk et al. . Moreovek et al. ,59–61. Tk et al. . Finallyk et al. .4-CONH2, 3--CONH2, Sal-3-CONH2 and Ac-Sal-3-CONH2 were strong inhibitors -CONH2 was the most potent with an IC50 of ~1.6 μM.We tested 18 aromatic foldamers  for inhihibitors A–4D. 2-CONH2, Sal-2-CONH2, 3-CONH2 and Ac-3-CONH2 failed to inhibit assembly 4-CONH2, 4-COMe, 4-COOH and 4-COβAla 3-CONH2, which possess three lysine side chains and four aromatic backbone units, were potent inhibitors 3--CONH2 were potent inhibitors, whereas 2---CONH2, 2---CONH2, --2-CONH2 and 2-CONH2 were ineffective assembly  and 3A. 4-COβAla  and 3A. hibitors  and 3A. ffective  and 3A.4-CONH2, 3--CONH2, Sal-3-CONH2 and Ac-Sal-3-CONH2 did not disassemble Aβ42 fibrils after 24 h (results not shown). Thus, these foldamers do not reverse Aβ42 fibrillization.Select small molecules that inhibit Aβ42 fibrillization also disassemble Aβ42 fibrils ,60. Howe4-CONH2, 3--CONH2, Sal-3-CONH2 and Ac-Sal-3-CONH2 did not inhibit NM fibrillization . Thus, (Arg-Benz)4-CONH2 does not stimulate NM fibrillization. Rather, we suggest that NM accesses a different prion strain in the presence of (Arg-Benz)4-CONH2. NM accesses different prion strains in the presence of certain small molecules, such as EGCG [Next, we assessed foldamer specificity by testing whether they inhibited amyloidogenesis of the prion domain, NM, of yeast Sup35 . 3--CONH2 and Ac-Sal-3-CONH2 did not inhibit FUS aggregation 3-CONH2 only inhibited seeded Aβ42 fibrillization when present at a 4-fold molar excess over Aβ42 3-CONH2 are more potent inhibitors of spontaneous Aβ42 fibrillization 3-CONH2 likely preferentially inhibit the rearrangement of Aβ42 oligomers into fibril-nucleating species [4-CONH2 and Sal-3-CONH2 have reduced ability to inhibit assembly.(Arg-Benz)ver Aβ42 E. Even alization A and 4C lization A and 6C. species . Once Aβ3-CONH2 and 3--CONH2 inhibited seeded Aβ42 fibrillization at all concentrations tested -CONH2 was more potent with an IC50 of ~2.5 μM 3--CONH2 inhibit Aβ42 fibrillization even after formation of species that nucleate fibrillization.Ac-Sal-s tested D and 6E.3-CONH2 and Ac-Sal-3-CONH2 did not block spontaneous Aβ43 fibrillization 3-CONH2. These findings suggest that potent inhibitors of spontaneous Aβ42 fibrillization may not inhibit spontaneous Aβ43 fibrillization. By contrast, (Arg-Benz)4-CONH2 and 3--CONH2 blocked spontaneous Aβ43 fibrillization 3--CONH2 was ~3.1 μM 3-CONH2 did not inhibit seeded Aβ43 fibrillization 3--CONH2 blocked seeded Aβ43 fibrillization 3--CONH2 against seeded Aβ43 fibrillization was ~1.7 μM.Aβ43 fibrils eliminated the lag phase of Aβ43 assembly 3--CONH2, Sal-3-CONH2 and Ac-Sal-3-CONH2 inhibited spontaneous and seeded Aβ42 fibrillization 3--CONH2 inhibited spontaneous and seeded Aβ43 fibrillization o effect . By contlization , whereaslization .Could foldamers inhibit the formation of toxic Aβ42 and Aβ43 oligomers? To assess toxic Aβ42 and Aβ43 oligomer formation, we employed the conformation-specific A11 antibody, which specifically recognizes preamyloid oligomers formed by multiple proteins, including Aβ42, but not monomers or fibrils . We asse4-CONH2 3-CONH2 and Ac-Sal-3-CONH2 had no effect on the abundance of A11-reactive Aβ42 or Aβ43 oligomers after 0.5 h 3-CONH2 and Ac-Sal-3-CONH2, A11-reactive Aβ42 species remained at higher levels and did not decline as much as they did in the absence of foldamer 3-CONH2, and Ac-Sal-3-CONH2 stabilize A11-reactive conformers. (Arg-Benz)4-CONH2, but not Sal-3-CONH2 or Ac-Sal-3-CONH2, had a similar effect on A11-reactive Aβ43 species 3-CONH2 4-CONH2 , had no 4-CONH2 A. (Arg-B species A. By con)3-CONH2 C and 7D.3--CONH2 inhibited the formation of A11-reactive Aβ42 and Aβ43 conformers after 0.5 h -CONH2 prevented further accumulation of A11-reactive Aβ42 and Aβ43 conformers -CONH2 inhibits fibrillization as well as toxic oligomer formation by Aβ42 and Aβ43. 3--CONH2 might inhibit Aβ42 and Aβ43 misfolding by a mechanism that is distinct to the other foldamers and arrests Aβ42 and Aβ43 misfolding prior to an A11-reactive oligomeric state.4-CONH2, had no effect on toxicity 3-CONH2 and Ac-Sal-3-CONH2 had no effect on the toxicity of Aβ42 conformers after 0.5 h of assembly 3-CONH2 and Ac-Sal-3-CONH2 inhibit spontaneous Aβ42 fibrillization such that more toxic conformers are maintained 3-CONH2 affected the toxicity of conformers after 0.5 h or 4 h -CONH2, which inhibited the formation of A11-reactive Aβ42 and Aβ43 conformers after 0.5 h -CONH2 was the only foldamer that antagonized Aβ42 and Aβ43 toxicity.3--CONH2 inhibits spontaneous and seeded Aβ42 and Aβ43 fibrillization and reduces accumulation of toxic Aβ42 and Aβ43 conformers. This combination of properties could have therapeutic potential for three reasons. First, 3--CONH2 antagonizes Aβ42 as well as Aβ43, which is an often overlooked but highly toxic Aβ species [3--CONH2 inhibits the formation of toxic Aβ42 and Aβ43 conformers, which could reduce localized neurodegeneration [3--CONH2 inhibits seeded Aβ42 and Aβ43 assembly, which could prevent the spreading of Aβ pathology throughout the brain in AD [3--CONH2 against Aβ misfolding and toxicity in the metazoan nervous system. species –16. Seconeration . Thirdlyin in AD –31. Furt38VVIAT43) of Aβ43.Future studies will reveal the mechanisms by which foldamers antagonize Aβ-misfolding. Foldamers have amides oriented appropriately  to blockAromatic foldamers could be useful amyloidogenesis inhibitors for various disease-associated proteins. Indeed, another class of aromatic foldamer inhibits amylin fibrillization, which is connected to Type 2 diabetes . Thus, f


The asymmetric unit of the title compound contains two independent molecules that are interconnected through an offset π–π interaction. The fused benzene and pyran-2-one rings in each molecule are essentially coplanar. Similarly, the coumarin ring system and the 7-propoxy substituent are close to being coplanar. 13H14O3, contains two independent molecules, A and B, that are interconnected through an offset π–π interaction [inter-centroid separation = 3.6087 (4) Å]. The fused benzene and pyran-2-one rings in each molecule are essentially coplanar, having dihedral angles of 1.22 (12) and 1.57 (12)° for molecules A and B, respectively. Similarly, the coumarin ring system and the 7-propoxy substituent are close to being coplanar . In the crystal, the molecules are connected by C—H⋯O hydrogen bonds, forming supramolecular tapes along [100] that are linked into a three-dimensional network by C—H⋯π interactions, as well as by the aforementioned π–π interactions.The asymmetric unit of the title compound, C H-1-benzopyran-2-one) is a plant-derived natural product known for its pharmacological properties such as anti-inflammatory, anticoagulant, antibacterial, antifungal, antiviral, anticancer, antihypertensive, antitubercular, anticonvulsant, anti-adipogenic, antihyperglycemic, anti-oxidant and neuroprotective properties. Dietary exposure to benzopyrones is significant as these compounds are found in vegetables, fruits, seeds, nuts, coffee, tea and wine . Bond lengths between equivalent non-H atoms of each molecule are similar, with differences less than 3 s.u.The asymmetric unit of (I)A; r.m.s. deviation = 0.0192 Å, dihedral angle between the two six-membered rings of 1.57 (12)° for B].The fused aryl and pyran-2-one rings in each molecule are individually planar (r.m.s. deviations < 0.0064 for aryl rings and < 0.0141 Å for pyran-2-one rings) and form single planar units   and potassium carbonate  were dissolved in acetone (2.0 ml) and kept at room temperature. After 20 minutes, n-propylbromide  was added drop wise and the reaction mixture was heated to reflux (313 K) and monitored by TLC. After completion of the reaction (six days), the reaction mixture was filtered and the solid residue was washed off with cold water (10 ml). The total mother liquors were concentrated under reduced pressure and then poured into water and extracted with ethyl acetate (3 × 15 ml). The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure to give a white-coloured solid (m.p. 347.55–348.35 K). Single crystals were obtained from methanol. 1H NMR data  d: 1.02 , 1.82 , 2.36 , 3.94 , 6.08 , 6.76 , 6.82 , 7.45 .The title compound was prepared by aryl = 0.95 Å, Uiso(Haryl) = 1.2 Ueq(C); C—Hmethylene = 0.99 Å, Uiso(Hmethylene) = 1.2 Ueq(C); C—Hmethyl = 0.98 Å, Uiso(Hmethyl) = 1.5 Ueq(C)].Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S1600536814023678/tk5346sup1.cifCrystal structure: contains datablock(s) I, New_Global_Publ_Block. DOI: 10.1107/S1600536814023678/tk5346Isup2.hklStructure factors: contains datablock(s) I. DOI: 1031270CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


TCRVγ/δ recombination in fetal thymus is lost in mTORC1KO thymus, leading to elevated γδT17 differentiation and rearranging of fetal specific TCRVγ/δ in adulthood. Thus, mTORC1 is central for TEC development/function and establishment of thymic environment for proper T cell development, and modulating mTORC1 activity can be a strategy for preventing thymic involution/atrophy.Thymus is crucial for generation of a diverse repertoire of T cells essential for adaptive immunity. Although thymic epithelial cells (TECs) are crucial for thymopoiesis and T cell generation, how TEC development and function are controlled is poorly understood. We report here that mTOR complex 1 (mTORC1) in TECs plays critical roles in thymopoiesis and thymus function. Acute deletion of mTORC1 in adult mice caused severe thymic involution. TEC-specific deficiency of mTORC1 (mTORC1KO) impaired TEC maturation and function such as decreased expression of thymotropic chemokines, decreased medullary TEC to cortical TEC ratios, and altered thymic architecture, leading to severe thymic atrophy, reduced recruitment of early thymic progenitors, and impaired development of virtually all T-cell lineages. Strikingly, temporal control of IL-17-producing γδT (γδT17) cell differentiation and  The thymus is essential for making T cells but undergoes age- or stress-associated atrophy. This study demonstrates that mTOR complex 1 in thymic epithelial cells is crucial for correct thymic architecture and the production of mature T cells. TCRVγ/δ recombination. Our results establish mTORC1 as a central regulator for TEC development/function and for the establishment of normal thymic environment for proper T cell development. We suggest modulating mTORC1 activity as a strategy for preventing thymic involution/atrophy.The thymus is the primary organ for T cell generation. Abnormal thymus function profoundly affects host immunity and numerous diseases. Thymopoiesis and thymus function rely on orchestrated interaction between multiple cell types representing different origins. Among them, thymic epithelial cells (TECs) are crucial for thymus development and maintenance and T cell generation. How TEC development and function are regulated is poorly understood. The mammalian/mechanistic target of rapamycin (mTOR), a serine/threonine kinase, signals with two complexes, mTORC1 and mTOC2, to control metabolism, growth, proliferation, and survival. Using a mouse model with mTORC1 selectively ablated in TECs, we demonstrate that mTORC1 in TECs plays critical roles in thymopoiesis and thymus function. Absence of mTORC1 results in impaired TEC maturation and function, altered thymic architecture, severe thymic atrophy, and impaired development of virtually all T-cell lineages. Moreover, it also causes increased generation of IL-17–producing γδT (γδT17) cells and fetal-specific γδT subsets in adult thymus, revealing that mTORC1 in TECs is central for temporal control of γδT17 differentiation and  The thymus is the primary organ for T cell development and generation of a diverse repertoire of T cells that are crucial for host defense but are also self-tolerated. Thymic epithelial cells (TECs) are essential for thymopoiesis and establish an environment that properly nurtures T cell development . TECs in−CD4−CD8− double negative (DN) cells that express CD44, cKit, and CD24 but not CD25, undergo maturation sequentially through the DN2, DN3, and DN4 stages [TCRVγ (Vγ5 and Vγ6) and TCRVδ1 segments recombine only in the fetal thymus [TCRVγ/Vδ usage has been unclear.During T cell ontogeny, early T cell progenitors (ETPs) enter into the thymus at the corticomedullary junction. Following initial migration toward the cortex, ETPs, which are Lin4 stages . TCRγδ T4 stages . Interes4 stages , 13. Addl thymus , 15. WheTCRβ may overcome the developmental checkpoint between DN3 and DN4 to reach the CD4+CD8+double positive (DP) stage and adopt the αβT fate. Expression of a functional TCRα/β that recognizes self-peptide-major histocompatibility complex (MHC) complexes presented by cTECs triggers positive selection for maturation to the CD4+CD8− or CD4−CD8+ single positive (SP) stage [DN thymocytes uncommitted to the γδT fate but with in-frame rearranged P) stage . After pP) stage . In the P) stage . Small sP) stage , 19, 20.iNKT cells, Tregs, and cαβT cells [iNKT cells, and γδT cells correlated with decreased recruitment of ETPs in the thymus. Moreover, fetal thymus restricted γδT17 differentiation and TCRVγ5/6Vδ1 recombination occur in adult thymus in the absence of mTORC1 in TECs, suggesting that TECs and thymic environment rather than hematopoietic stem cells confer temporal control of γδT cell development.The serine/threonine kinase mammalian/mechanistic target of rapamycin (mTOR) has the ability to integrate various environmental and intracellular stimuli and cues to control cell growth, proliferation, survival, autophagy, and metabolism. Mammalian/mechanistic target of rapamycin complex 1 (mTORC1), one of the two complexes, which contains a crucial and unique adaptor molecule Raptor, phosphorylates multiple substrates such as S6K1 and 4E-BP1 to promote protein, nucleic acid, and lipid synthesis, which is crucial for cell growth and proliferation . mTOR isβT cells –28. HoweWe first examined S6 phosphorylation, an mTORC1/S6K1 dependent event, in TECs from mice aged at 9 d, 3 wk, and 10 wk. S6 phosphorylation was the strongest in TECs from 9-d-old mice but the lowest in cells from 10-wk-old adult mice . FurtherRptorf/f-Rosa26-ERCre (Rptf/f-ERCre or eKO) and control Rptorf/f  mice following tamoxifen injections on days 1, 2, and 5. On day 8, thymi in Rptf/f-ERCre mice were much smaller than Rptf/f mice  staining of thymus thin sections from tamoxifen-treated  medulla , which w medulla . The shrd 6-fold , their td 6-fold . The pre numbers . Thus, aRptf/f-LckCre or Rptf/f-CD4Cre mice did not cause obvious thymic atrophy or abnormal distribution of DN, DP, and SP populations in the thymus [Rptf/f-Foxn1Cre  and Rptf/f (WT) mice. Foxn1Cre mice contain an IRES-Cre cassette inserted into the 3’ untranslated region in the Foxn1 locus to direct Cre expression starting on embryonic day 11.5 in TECs [Rptf/f-Foxn1Cre mice displayed apparent thymic atrophy , resulting in substantial decreases of mTEC/cTEC ratios after 3 wk of age  cells are also generated in the thymus. Unlike cαβT cells, they are positively selected after engagement of the iVα14TCR with self-lipid ligand-CD1d complex expressed on DP thymocytes [iNKT cell development is largely unclear. The percentages of PBS57-loaded CD1d-Tetramer (CD1Dtet)+ TCRβ+iNKT cells were decreased in the thymus of 10-d- and 6-wk-old Rptf/f-Foxn1Cre mice , 2 (CD24−CD44+NK1.1−), and 3 (CD24−CD44+NK1.1+) iNKT cells in these mice were similar to WT controls , DN2 (cKit+CD44+CD25+), DN3 (CD44−CD25+), and DN4 (CD44−CD25−) subsets within Rptf/f-Foxn1Cre Lin− thymocytes did not deviate greatly from E16 fetus, 3-wk- and 6-wk-old mice in the control group  yet to be defined.Using xpansion . Second,Rptf/f-ERCre mice, we have also shown that acute deletion of mTORC1 in adult mice quickly causes severe thymic atrophy, altered thymic architecture, and substantial reduction of DP thymocytes within one week. Our data are consistent with previous observations that rapamycin treatment induces thymic atrophy and DP thymocyte death in mice [Raptor does not affect thymus size and total cellularity [Rptf/f-ERCre mice after tamoxifen injection, our results do not rule out a potential role of mTORC1 for mature c/mTEC homeostasis after prolonged deletion. It is important to point out that a recent study has found severe thymic atrophy in the same strain of mice 2–3 wk after tamoxifen injection and has attributed thymic atrophy to early T cell developmental blockade [Raptor in mature TECs is required.Using  in mice . Since Tlularity , we propblockade . TECs anblockade . NeverthAn important question that remains to be addressed is how mTORC1 controls TEC development and function. The transcription factor Foxn1 is essential for TEC development and thymopoiesis , 19 as wRptf/f-Foxn1Cre mice, individual T cell lineages appear to display differential sensitivities to mTORC1 deficiency in TECs. iNKT cells appear most stringently dependent on mTORC1 signaling in TECs, which is surprising, because their positive selection relies on engagement of the iVα14TCR with self-lipid ligands presented by CD1d expressed on DP thymocytes in the cortex rather than TECs [Vα14-Jα18 recombination in DP thymocytes were not obviously affected in Rptf/f-Foxn1Cre mice  to promote early iNKT cell development. Similar to iNKT cells, nTregs are more sensitive than cαβT cells to mTORC1 deficiency. nTreg differentiation depends on self-peptide-MHC-II presented by or derived from mTECs [Rptf/f-Foxn1Cre thymus may contribute to a more severe impairment of nTreg generation. Furthermore, a thymic environment such as local TGFβ and CD80/86-mediated costimulation modulates nTreg generation [Rptf/f-Foxn1Cre mice could also contribute to nTreg deficiency.Although the generation of virtually all T cell lineages is impaired in han TECs . CD1d exCre mice . A recenelopment . In Rptom mTECs , 4. The neration , 47. AltTCRVγ5, Vγ6, and Vδ1 recombination, which predominantly or strictly occur in fetal thymus and are switched off in adult thymus [Vγ5/6/Vδ1, leading to uncontrolled γδT17-cell generation and Vγ5/6Vδ1 recombination in adulthood. Our data suggest that mTORC1 controls TECs to enforce such temporal Vγ5/6/Vδ1 recombination and γδT17 generation. Interestingly, a recent report has also found impaired temporal control of γδT17 differentiation and Vγ/Vδ recombination in adult β5t mutant mice [Vγ5/6/Vδ1 usages in an in vitro culture system [Rptf/f-Foxn1Cre mice, it is possible that mTORC1 may play a crucial role in cTECs to restrain Vγ6+ γδT17 cell generation in adult thymi.Immune cells undergo specific switches during development . Among tt thymus –15. Mechant mice . Thus, ae system , our date system  suggest e system . BecauseVγ5/6Vδ1chromatin for recombination. If this is true, mTORC1 may play an important role in the transition of TECs from fetal stage, which may be young and permissive for γδT17 differentiation and TCRVγ5/6/Vδ1 recombination, to adult TECs, which are aged and impermissive for γδT17 differentiation and TCRVγ5/6/Vδ1 recombination. γδT17 differentiation requires transcription factor RORγt and signals from Notch, TGFβ, and LTβR but not TCR [Vγ5/6Vδ1 recombination in an mTORC1-dependent manner.Important issues to be addressed in the future are how specific determinants in TECs dictate temporal control of γδT cell development and how mTORC1 signaling impact on these determinants. A potential possibility is that fetal and adult TECs are qualitatively different in a way that only fetal TECs confer an environment suitable for γδT17 differentiation and for opening  not TCR , 51, 52  not TCR . WhetherDespite its importance, thymus undergoes involution or atrophy with advanced age or under certain pathological conditions. Thymic involution leads to a decrease in T cell production and shrinking of the T cell repertoire, which can result in impairment of adaptive immunity and propensity for autoimmunity , 55. Alt2 for experiments.Mouse experiments described were approved by the Institutional Animal Care and Use Committee of Duke University. Mice were euthanized with CORptorf/f mice [Foxn1Cre mice [Rptf/f-Rosa26-ERCre mice were previously reported [f/f mice  were purCre mice  were kinreported , 58. ForTECs were prepared according to a published protocol with modifications . In brieTotal lung cells were isolated according to a published protocol with modifications. Brieflyb (clone B21.5), β11 (clone RR3-15), β12 (clone MR11-1), β13 (clone MR12-3), β14 (clone 14–2), and β17a (clone KJ23) were purchased from BD Pharmingen. The 17D1 (anti-Vγ5Vδ1) monoclonal antibody was prepared from cultured hybridoma supernatant after ammonium sulfate precipitation and affinity purification with a goat anti-rat IgM (Jackson ImmunoResearch Laboratories) conjugated Sepharose-4B beads (Amersham Pharmacia Biotech AB) and was conjugated with biotin (ProteoChem) according to a manufacturer’s protocol. The 17D1 antibody detects Vγ6Vδ1 when pretreated with the anti-TCRγδ (GL3) antibody [http://zenodo.org/record/34843 or DOI URL: http://dx.doi.org/10.5281/zenodo.34843).Fluorochrome-conjugated anti-CD45.2 (clone 104), CD45(clone 30-F11), CD45.1(clone A20), EpCAM/CD326 (clone G8.8), Ly51 (clone 6C3), MHCⅡ-I-A/I-E (clone M5/114.15.2), CD40 (clone 3/23), CD4 (clone GK1.5), CD8 (clone 53–6.7), TCR-β (clone H57-597), TCRγδ , CD44 (clone IM7), CD24 (clone M1/69), CD25 (clone PC61.5), CD27 (clone LG.3A10), c-Kit/CD117 (clone 2B8), CD62L (clone MEL-14),Gr1 (clone RB6-8C5), CD11b (clone M170), CD11c (clone N418), F4/80 (clone BM8), CD1d (clone 1B1), CD1d tetramer (NIH tetramer facility), B220 (clone RA3-6B2), CD19 (clone 6D5), TER119/Erythroid Cells (clone TER-119), CD3e (clone 145-2C11), Annexin-V, Streptavidin,CCR6 (clone 29-2L17), IFN-γ (clone XMG1.2), IL-17A (clone TC11-18 H10.1), Foxp3 , Anti-TCR-Vγ1.1 , Vγ4 , Vγ5 (clone 536), Vγ7 (clone F2.67), and Vδ6.3  were purchased from Biolegend unless indicated otherwise. Ulex Europaeus Agglutinin I  was purchased from vector laboratories. Phospho-S6  antibody was from Cell Signal Technology. FITC-conjugated TCR-Vβ usage kit, including anti-TCRβ2 (clone B20.6), β3 (clone KJ25), β4 (clone KT4), β5.1/5.2 (clone MR9-4), β6 (clone RR4-7), β7 (clone TR310), β8.1/8.2 (clone MR5-2), β8.3 (clone IB3.3), β9 (clone MR10-2), β10Thymocytes and single cell suspensions from other organs were stimulated with phorbol 12-myrustate 13-acetatae  plus ionomycin (500 ng/ml) in the presence of GolgiPlug (1 ng/ml) for 4 h. After cell surface staining, intracellular staining for IL-17A and IFN-γ was performed by using the BD Biosciences Cytofix/Cytoperm and Perm/Wash solutions.For thymocytes, mice were i. p. injected with 5-bromo-2-deoxyuridine  and were stained 4 h after injection to assess BrdU incorporation. For embryonic ETPs, mice pregnant for 16 d were i. p. injected with 3 mg BrdU, and fetal thymi were collected 6 h after injection to assess BrdU incorporation. For TECs, mice were i. p. injected with 1 mg BrdU 3 times every 24 h, and thymi were collected 14 h after the last injection for TEC preparation. After cell surface staining, cells were intracellularly stained for BrdU using a BrdU Flow Kit (BD Biosciences) according to the manufacturer’s protocol.Thymus for H&E staining were fixed in 10% formalin solution for 1 d, and then changed into 70% ethanol. Paraffin-thin sections were stained with H&E according to standard protocols. Thymus for immunofluorescent staining were embedded in OCT  and frozen immediately in −80°C. Frozen thin sections (5 μm) were fixed in a 1:1 mixture of acetone and methanol at −20°C for 8 min. After blocking with PBS containing 3% BSA with 0.1% Tritonx-100 for 30–45 min at room temperature (RT), samples were stained using primary rat-anti-mouse-keratin 8  and rabbit-anti-mouse-keratin 5 , followed by secondary Rhodamine-conjugated-donkey anti-rabbit IgG (1:400 dilution) and FITC-conjugated-goat anti-rat IgG . Samples were mounted with Vector mounting solution containing DAPI (Vector) and allowed to dry overnight at RT or 4°C in the dark before imaging. Images were acquired using a Zeiss ApoTome Microscope and analyzed using PhotoshopCS4 software.+CD45.2+ WT bone marrow cells were depleted off T cells using a PE-conjugated anti-CD3 antibody, anti-PE-antibody conjugated magnetic beads, and LD columns (Miltenyi Biotec) according to the manufacturer’s protocol. CD45.2+Rptf/f and Rptf/f-Foxn1Cre mice were lethally irradiated (1000 rad) and were intravenously injected with 1.0 x 107 T cell depleted bone marrow cells 4 h after irradiation. Recipient mice were analyzed 5–6 wk later.CD45.1Rptf/f-Foxn1Cre and WT littermates were plated at 1 x 107 cells/well in 96-well U-bottom plates and treated with or without fluorescent 100 μM 2-Amino)-2-Deoxyglucose  in PBS and incubated at 37°C with 5% CO2 for 30 min. The 2-NBDG uptake reaction was stopped by removing culture medium and washed with pre-clod PBS two times. Cells were stained for surface molecules before analysis with flow cytometry.Single cell suspension made for TEC preparations from 3-wk-old +CD45−UEA-1+Ly51−) and cTECs (Epcam+CD45−UEA-1+Ly51−) using the Trizol reagent (Sigma). The first strand cDNAs were reversely transcribed using an iScript cDNA Synthesis Kit (Bio-Rad). Quantitative real-time PCR (qRT-PCR) was performed using a Mastercycler Realplex (Eppendorf) and the SensiMix SYBR No-ROX Kit (Bioline). Data were analyzed using the 2-Ct method after normalization to β-actin expression and shown as relative expression levels. For semi-quantitative PCR, cDNA template in 1:4 serial dilutions from each sample was used. For Vα14 to Jα2, Jα18, and Jα56 recombination, genomic DNA isolated from sorted CD4+CD8+ DP thymocytes from Rptf/f and Rptf/f-Foxn1Cre mice was used as the template for semi-quantitative PCR according to a previously published protocol except that TSC1 was used as loading control [Total RNAs were extracted from E16 thymi or sorted 10 d-old mTECs Click here for additional data file.S1 FigA. Gating strategies to get to TECs for B. Gating strategies to get to live thymocytes for C. Gating strategies to get to thymocytes for D. Gating strategies to get to TECs for (PDF)Click here for additional data file.S2 FigGating strategies to get to TECs for (PDF)Click here for additional data file.S3 FigFoxn1Cre+ and Foxn1Cre− 3-wk-old litter-mates were examined. A. Thymus size. B. Total thymic cellularity. Each circle or square represents one Foxn1Cre and WT, respectively. Bars represent mean ± SEM. C. Representative dot plots of thymocytes. D. Percentages and numbers of thymocyte subsets. E. Representative dot plots of TECs. F. Percentages and numbers EpCAM+CD45− TECs in thymus. G. Percentages and numbers of mTECs and cTECs. H. MHCII and CD40 staining of gated mTECs. Data shown represent three experiments .(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 FigA. Gating strategy for B. Gating strategy for (PDF)Click here for additional data file.S6 FigRptf/f and Rptf/f-Foxn1Cre mice were stained with CD4, CD8, and individual TCRVβ chains using a TCRβ staining kit (BD Biosciences). Bar graphs represent mean ± SEM of individual TCRβ chain percentages in gated CD4 or CD8 T cells.Splenocytes from (PDF)Click here for additional data file.S7 FigA. Gating strategy for B. Gating strategy for C. Gating strategy for (PDF)Click here for additional data file.S8 FigA. Gating strategy for B. Gating strategy for (PDF)Click here for additional data file.S9 FigA. Gating strategy for B. Gating strategy for (PDF)Click here for additional data file.S10 FigFoxn1Cre+ and Foxn1Cre− 3-wk-old litter-mates were examined. A. γδT percentages and numbers in the thymus. B. Representative dot plots of IL-17A and IFNγ staining in thymic γδT cells. Thymocytes were stimulated with PMA plus ionomycin in the presence of brefeldin A (BFA) for 4 h followed by cell surface and intracellular staining. Dot plots show IL-17A and IFNγ expression in gated TCRγδ+TCRβ− cells. C. γδT1 and γδT17 percentages and numbers in the thymus. D. γδT1 and γδT17 numbers in the thymus. Data shown represent three experiments .(PDF)Click here for additional data file.S11 FigA. Sorting strategy for γδT cells used in B. Gating strategy for C. Gating strategy for (PDF)Click here for additional data file.S12 FigData shown represent three experiments.(PDF)Click here for additional data file.S13 FigA. Gating strategy for B. Gating strategy for C. Gating strategy for D. Gating strategy for (PDF)Click here for additional data file.S14 FigA. Gating strategy for B. Gating strategy for (PDF)Click here for additional data file.S15 FigRelative Foxn1 mRNA levels in sorted TECs from 10-d-old mice were determined by real-time qPCR.(PDF)Click here for additional data file.S16 FigA. Genomic DNA isolated from sorted CD4+CD8+ DP thymocytes from Rptf/f and Rptf/f-Foxn1Cre mice were utilized for detection of Vα14 to Jα2, Jα18, and Jα56 recombination using semi-quantitative PCR. TSC1 was used as loading control. B. Overlaid histograms show CD1d expression on DP thymocytes. Data shown represent three experiments.(PDF)Click here for additional data file.


E conformation about the C=N bonds and in each molecule there is an intramolecular O—H⋯N hydrogen bond, forming an S(6) ring motif. In the crystals of both compounds, zigzag chains are formed via N—H⋯N hydrogen bonds, in the  and adopt an E conformation with respect to the double bond of the hydrazone bridge. In each molecule, there is an intramolecular O—H⋯N hydrogen bond forming an S(6) ring motif. The dihedral angles between the mean planes of the isonicotinoyl ring and the cresol ring in (I) or the fluorophenol ring in (II) are 10.49 (6) and 9.43 (6)°, respectively. In the crystals of both compounds, zigzag chains are formed via N—H⋯N hydrogen bonds, in the [10-1] direction for (I) and [010] for (II). In (I), the chains are linked by weak C—H⋯π and π–π stacking interactions , leading to the formation of a three-dimensional supramolecular architecture. In (II), adjacent chains are connected through C—H⋯O hydrogen bonds to form sheets parallel to (100), which enclose R44(30) ring motifs. The sheets are linked by weak C—H⋯π and π–π  interactions, forming a three-dimensional supramolecular architecture.Two derivatives of the well-known iron chelator, ( Both compounds have an E conformation with respect to the double bond of the hydrazone bridge (C7=N3) with the C8—C7=N3—N2 torsion angles being −179.03 (12) and −177.61 (11)° for (I)et al., 2001abet al., 2010et al., 2014.The molecular structures of the title compounds, (I)via N—H⋯N hydrogen bonds -N-(2-hydroxybezylydene)isonicotinohydrazide substructure. They include the isotypic crystal structures with chloride A solution of isonicotinic acid hydrazide  and the appropriately substituted salicyl aldehyde (1.47 mmol) in a mixture of ethanol (3 ml) and water (1 ml) containing a catalytic amount of acetic acid was heated to reflux for 5 h. The reaction mixture was allowed to cool to room temperature, resulting in the formation of a white precipitate. The reaction mixture was filtered and the isolated solid was washed with diethyl ether and dried 1H NMR  d 2.25 , 6.84 , 7.12 , 7.40 , 7.84 , 8.63 , 8.79 , 10.82 , 12.26 . HR–MS (ES+) C14H14N3O2 requires 256.1086 [M+H]+; found 256.1051.Spectroscopic data for (I)1H NMR  d 6.94 , 7.16 , 7.46 , 7.84 , 8.67 , 8.80 , 10.84 , 12.35 . HR–MS (ES+) C13H11FN3O2 requires 260.0835 [M+H]+; found 260.0831.Spectroscopic data for (II)Uiso(H) = 1.5Ueq and 1.2Ueq for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989016009762/su5301sup1.cifCrystal structure: contains datablock(s) Global, I, II. DOI: 10.1107/S2056989016009762/su5301Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016009762/su5301Isup4.cdxSupporting information file. DOI: 10.1107/S2056989016009762/su5301IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989016009762/su5301Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016009762/su5301IIsup6.cmlSupporting information file. DOI: 1485834, 1485833CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Each molecule forms four N—H⋯N hydrogen bonds with two neighbouring molecules to constitute a one-dimensional ladder-like structure along the  8H8N6, two imidazolyl groups are separated by a zigzag –CH=N—N=CH– linkage. An inversion center is located at the mid-point of the N—N single bond and the complete molecule is generated by symmetry. In the crystal, each molecule forms four N—H⋯N hydrogen bonds with two neighbouring molecules to constitute a one-dimensional ladder-like structure propagating along the a-axis direction.In the title compound, C In particular, hydrogen bonding, which is a powerful organizing force in designing a variety of supramolecular and solid-state architectures methylene]hydrazine (I)via hydrogen bonds. The functionality of molecule (I)Imidazoles, containing two nitrogen atoms, possess both hydrogen-bond donating and accepting sites and are superior building blocks for supramolecular architectures. Many imidazole-containing polydentate ligands derived from hydrazine find a wide range of applications in coordination chemistry owing to their chelating ability (Zhou i = 5.937 (3) Å [the distance between the centroids of the imidazolyl groups is 8.103 (3) Å]. The molecule possesses an inversion center located in the mid-point of the N—N single bond and the complete molecule is generated by symmetry. The molecule appears in a Z(EE)Z configuration and its geometry is similar to that of 1,2-bis[(1H-imidazol-5-yl)methylene]hydrazine  Å; symmetry code: (i) –x, −y + 1, −z + 2] , C=N double bond [C6—N7 = 1.2795 (19) Å] and C=N—N bond angle [C6=N7—N7i = 111.41 (15)°], are comparable to the corresponding parameters found in 1,4-bis(3-pyridyl)-2,3-diaza-1,3-butadiene hydrazine  of imidazole-2-carboxaldehyde  was added to a methanol solution (10 mL) of hydrazine monohydrate . The mixture was stirred for 3 h and the precipitate was collected by filtration. Single crystals suitable for X-ray diffraction studies were obtained by diffusion of diethyl ether into a DMSO solution of the title compound (I)Uiso were refined, giving an N—H distance of 0.95 (2) Å. The C-bound H atoms were subsequently treated as riding atoms in geometrically idealized positions: C—H distances of 0.95 Å with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016004497/bg2582sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016004497/bg2582Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016004497/bg2582Isup3.cmlSupporting information file. DOI: 1468833CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The coordination complex exhibits an inversion centre through the CH 2(C8H8O8)(C18H15P)4]·3CH2Cl2, crystallizes with one and a half molecules of dichloromethane in the asymmetric unit. The coordination complex exhibits an inversion centre through the central CH2—CH2 bond. The AgI atom has a distorted trigonal–planar P2O coordination environment. The packing is characterized by intermolecular T-shaped π–π interactions between the phenyl rings of the PPh3 substituents in neighbouring molecules, forming a ladder-type superstructure parallel to [010]. These ladders are arranged in layers parallel to (101). Intramolecular hydrogen bonds between the OH group and one O atom of the Ag-bonded carboxylate group results in an asymmetric bidendate coordination of the carboxylate moiety to the AgI ion.The molecular structure of the tetrakis(triphenylphosphanyl)disilver salt of butane-1,1,4,4-tetracarboxylic acid, [Ag The weak interaction to the O2 atom occurs below this AgP2 plane with an interaction to the CO2 group of 67.38 (17)° with, however, two nearly equal C1—O1/O2 bond lengths of 1.251 (3) (O1) and 1.261 (3) Å (O2). Both phosphanes reveal an eclipsed conformation regarding the phenyl rings of 2.09 (10)°.The Ag1 atom exhibits a somewhat distorted trigonal–planar PT-shaped π–π interactions S, as the hydrogen-bond donor, to the hydroxyl O3 atom .One dichloromethane is stabilized by a non-classical hydrogen-bridge bond from C1m Table 1, which aFurther intermolecular interactions involving hydrogen bonds or O⋯Ag interactions are not observed.T-shaped trigonal fashion :tert-butanolate  was added to a solution of butane-1,1,4,4-tetracarboxyl acid  in 5 mL of tetrahydrofuran. After stirring overnight at ambient temperature, the reaction mixture was filtered off and the residue was washed trice with tetrahydrofuran (10 mL each) and dried under vacuum (yield 243 mg). Subsequently, the obtained colorless solid  was dissolved in water (15 mL) and added dropwise to a solution of silver nitrate  in water (8 mL). After 12 h of stirring the colorless precipitate was filtered off and washed twice with water (10 mL each) and dried in a desiccator. The desired colorless butane-1,4-dicarboxyl-1,4-dicarboxylatodisilver(I) was obtained in a yield of 71%, based on butane-1,1,4,4-tetracarboxyl acid . Analysis calculated for C8H8Ag2O8 (447.88): C, 21.45; H, 1.80. Found: C, 21.49; H, 1.68. IR : ν = 2977 (m), 2903 (m), 2461 (m), 1660 (s), 1549 (vs), 1380 (s), 1257 (s), 1069 (s), 955 (m), 711 (m).Potassium Synthesis of bis[bis(triphenylphosphane)silver(I)]:3  was added in a single portion at ambient temperature. After 12 h of stirring the reaction mixture was filtered through a pad of celite. After removal of all volatiles under reduced pressure, the title compound (I)1H NMR : δ = 7.39–7.29 , 2.93 , 2.04 . 13C{1H} NMR : δ = 175.9 , 134.0 , 131.7 , 130.6 , 129.1 , 50.8 , 29.6 . 31P{1H} NMR : δ = 10.3 (s). IR : ν = 3108 (w), 2993 (w), 1751 (s), 1494 (vs), 1106 (s), 752 (vs), 701 (vs).To a suspension of butane-1,4-dicarboxyl-1,4-dicarboxylatodisilver(I)  in 10 mL of tetrahydrofuran, PPhUiso(H) = 1.2Ueq(C) and a C—H distance of 0.93 Å for aromatic (AFIX 43), 0.98 Å for methine (AFIX 13) and 0.97 Å for methylene H atoms (AFIX 23). The same applies for the O-bonded H atom; however, the torsion angle was derived from electron density (AFIX 147). The structure contains three molecules of dichloromethane as the solvent. Both crystallographically independent molecules consist of two moieties each. One molecule was refined as disordered over two positions  with occupancies of 92.7 (2) and 7.3 (3)%, respectively. The less prevalent moiety of C1SB is located close to a crystallographic inversion centre and symmetry-related pairs are mutually exclusive. The second disordered molecule is located directly atop of another inversion centre with an occupancy of 0.5  I, 1R. DOI: 10.1107/S2056989016000797/zl2653Isup2.hklStructure factors: contains datablock(s) I. DOI: 1447419CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Chiral separation of 1 gave two enantiomers (+)−1 and (−)−1. The structure of 1 was established by comprehensive spectroscopic analysis and single crystal X-ray diffraction. The absolute configurations of enantiomers were determined by quantum mechanical calculation. Compound (+)−1 exhibited a better neurotrophic activity than racemate 1 by promoting nerve growth factor (NGF) induced PC12 cell neurite outgrowth, while (−)−1 showed a distinctive inhibitory effect. Furthermore, a mechanism study indicated that the two enantiomers influenced NGF-induced neurite outgrowth of PC12 cells possibly by interacting with the trkA receptor, and extracellular signal regulated kinases 1/2 (ERK1/2) and mitogen-activated protein kinase (MEK) in Ras/ERK signal cascade. But the phosphorylation level of serine/threonine kinase Akt1 and Akt2 in PI3K/Akt signal pathway showed no significant difference between (+)−1 and (−)−1.Merrillianoid ( Nerve growth factor (NGF) is the first member of neurotrophin, it acts on sympathetic and sensory neurons and functions as a trophic factor for distinct populations of neurons in central nervous system5NTR)NTR induces apoptosis in some neuron cells. Evidence suggests that NGF binding to TrkA leads to the activation of Ras/ERK and PI3K/Akt downstream pathways10The functions of NGF are mediated by high-affinity tyrosine kinase receptor type 1 (TrkA) and the low-affinity non-enzymatic p75 neurotrophin receptor -abeo-9,11-seco abietane diterpenoid and six other abietane diterpenoidsNatural products are rich sources of lead compounds for drug discovery due to their unique structure and bioactivities1), a racemic neolignan possessing unique benzo-2,7-dioxabicyclo[3.2.1]octane moiety was isolated from the branches and leaves of I. merrillianum, and chiral separation of 1 gave two enantiomers (+)−1 and (−)−1 −1 showed quite distinctive inhibitory effect also at concentrations as low as 1 μM. Moreover, a mechanism study indicated that the two enantiomers exerted their promoting or inhibtory activities possibly by regulating the phosphorylation of TrkA receptor, the extracellular signal regulated kinases 1/2 (ERK1/2), and mitogen-activated protein kinase (MEK) in Ras/ERK pathway. However the phosphorylation level of serine/threonine kinase Akt1 and Akt2 in PI3K/Akt pathway showed no significant difference between (+)−1 and (−)−1. These findings provided a pair of interesting neolignans for NGF-agonist and antagonist drug design, and revealed that absolute configurations dramatically affected biological activities of compounds.In this study, merrillianoid (nd (−)−1 . Biologi1 was obtained as colorless needle crystal. The IR spectrum displayed absorptions due to the C−O bands at 1033, 1203 cm−1, and the phenyl groups at 1464, 1508 cm−1. The HRESIMS spectrum gave a pseudo-molecular-ion peak [M + H]+ at m/z = 417.1915 , which corresponding to a molecular formula C23H29O7 with ten degrees of unsaturation.Compound 1H NMR spectrum of 1 displayed four singlet aromatic protons at δH = 6.39, 6.45, 6.46, 6.86 ppm, and one methyl and five methoxyl groups at δH = 1.65, 3.55, 3.74, 3.77, 3.83, 3.85 ppm. Furthermore, the −CHCH3 fragment was identified owing to the doublet methyl resonance at δH = 1.36 ppm  and the quartet proton resonance at δH = 4.49 ppm . An interpretation of the 13C and DEPT NMR spectra of 1 revealed 22 signals attributable to 23 carbon atoms, including nine quaternary carbon atoms , seven methine groups , and seven methyl groups . The above 1H, 13C and DEPT NMR spectroscopic data suggested that there were two 1,2,4,5-tetrasubstituted phenyl groups in 1, and some of the resonances were quite similar to those of lignanoids.The 1 came from the interpretation of 1H-1H COSY, HSQC, HMBC, and NOESY spectra (3 (δH = 3.83 ppm) to C-2 (δC = 147.9 ppm), 4-OCH3 (δH = 3.85 ppm) to C-4 (δC = 152.3 ppm), and 5-OCH3 (δH = 3.55 ppm) to C-5 (δC = 142.5 ppm), confirmed the primary locations of three methoxy groups. In the NOESY spectrum of 1, the correlations between 3-H (δH = 6.46 ppm) and 2-OCH3, 4-OCH3, 6-H (δH = 6.86 ppm) and 5-OCH3, 4-OCH3 and 5-OCH3 were observed. The above NOESY correlations, together with the key HMBC correlations from 3-H and 6-H to C-1 (δC = 115.2), C-2, C-4, and C-5, confirmed the presence of the first 1,2,4,5-tetrasubstituted phenyl group in 1. Furthermore the HMBC correlations of 7-H (δH = 3.82 ppm) with C-1, C-2, and C-6 (δC = 112.6 ppm), and 9-CH3 (δH = 1.65 ppm) with C-7 (δC = 38.9 ppm) and C-8 (δC = 106.0 ppm) assigned the C7-C8-C9 side chain to C-1, establishing phenyl propane unit A. The HMBC correlations from 4′-OCH3 (δH = 3.77 ppm) to C-4′ (δC = 148.9 ppm), and 5′-OCH3 (δH = 3.74 ppm) to C-5′ (δC = 143.1 ppm), confirmed the primary locations of another two methoxy groups. The NOESY correlations between 3′-H (δH = 6.45 ppm) and 4′-OCH3, 6′-H (δH = 6.39 ppm) and 5′-OCH3, 4′-OCH3 and 5′-OCH3 were observed, the above correlations, together with the HMBC correlations from 3′-H and 6′-H to C-1′ (δC = 117.9 ppm), C-2′ (δC = 146.5 ppm), C-4′ and C-5′ confirmed the presence of another 1′,2′,4′,5′-tetrasubstituted phenyl group in 1. The HMBC correlations of 7′-H (δH = 3.03 ppm) with C-1′, C-2′, and C-6′ (δC = 110.9 ppm), and 9′-CH3 (δH = 1.36 ppm) with C-7′ (δC = 47.9 ppm) and C-8′ (δC = 85.2 ppm) assigned the C7′-C8′-C9′ side chain to C-1′, and established phenyl propane unit B. A spin coupling system H-7/H-7′ was established by the 1H-1H COSY spectrum, which indicated that phenyl propane units A and B were connected by C(7′)-C(7) bond. The HMBC cross-peak from 8′-H (δH = 4.49 ppm) to C-8, suggesting the existence of an ether bridge between C-8′ and C-8, forming a tetrahydrofuran ring. Deducting nine degrees of unsaturation accounted for two 1,2,4,5-tetrasubstituted phenyl groups and one tetrahydrofuran ring, the remaining one degree of unsaturation suggested that an additional ring was required. On the basis of the chemical shift, C-8 was supposed to link to C-2′ through an oxygen atom. Thus, the structure of 1 was established as depicted, and named merrillianoid, which possessed unique structure featured with a ketal formed between C-8 and C-2′, C-8′, forming a characteristic benzo-2,7-dioxabicyclo[3.2.1]octane moiety , the correlations of 9′-CH3 with 7-H, and 9-CH3 with 6-H established the relative configuration of 1. The single crystal X-ray diffraction  further confirmed the relative configuration of 1 −1 and (−)−1, which had opposite optical rotation and Cotton effects (see In the NOESY spectrum of merrillianoid (ion of 1 . Howeverects see . However1 and (−)−1 with the calculated ECD spectra obtained by quatum mechanical computation with the Gaussian03 software ] ). Because of the rigid structure of 1 and the relative configuration established by NOESY experiment and single crystal X-ray diffraction, the arbitrarily assigned absolute configuration of 7R,8S,7′S,8′S for (+)−1 was geometrically optimized by using density functional theory (DFT) at B3LYP/3-21G level to afford three preffered conformers A–C (ΔE < 5.0 kJ/mol) -1 matched well with the experimentally measured CD spectrum of (+)−1, which showed negative Cotton effects at 190–200 nm, and positive Cotton effects at 204–218 nm. Thus the absolute configuration of (+)−1 was assigned as 7R,8S,7′S,8′S. Similarly, the absolute configuration of (−)−1 was determined to be 7S,8R,7′R,8′R by the same method as (+)−1  has been successfully applied to determine the absolute configurations of natural products19 kJ/mol) . The ECD+)−1 see .1 and (−)−1, and racemate 1 were evaluated using rat pheochromocytoma (PC12) cells as a model system of neuronal differentiation1 significantly increased PC12 cell differentiation rates , comparing with 20 ng/mL NGF alone . Enantiomer (+)−1 had a better promoting effect than racemate 1 with differentiation rates of 28.05 ± 0.71% at 1 μM, 35.47 ± 1.40% at 10 μM (P < 0.01), while (−)−1 exhibited a distinctive inhibitory effect with neuronal differentiation rates of 17.05 ± 0.71% at 1 μM, and 14.47 ± 1.40% at 10 μM (P < 0.01). Overall, despite having the same planar structure, the opposite absolute configurations for (+)−1 and (−)−1 resulted in distinctive effects on NGF-induced PC12 cell differentiation.The NGF-mediated neurotrophic activity of enantiomers (+)−1 and (+)−1 both increased significantly comparing to 20 ng/mL NGF , and the phosphorylation level of (+)−1 at 10 μM  was higher than that of racemate 1 . The phosphorylation level of (+)−1 and 1 was in proportion to their neurite outgrowth promoting activities. As for (−)−1, the relative ratio of p-TrkA, and p-MEK decreased dramatically  comparing with 20 ng/mL NGF. In a word, compound (+)−1 promoted the NGF-induced neurite outgrowth of PC12 cells by enhancing the phosphorylation level of TrkA receptor, and MEK, ERK1/2 in Ras/ERK signaling pathway, while (−)−1 inhibited neurite outgrowth of PC12 cells via suppressing the phosphorylation of the above proteins.We further investigated the possible molecular mechanism of merrillianoids on NGF-induced PC12 cell differentiation by western blotting analysisThe PI3K/Akt signaling cascade regulated cell survival and suppress apoptosis in a wide range of neuronal cell types10241 and (−)−1 with characteristic benzo-2,7-dioxabicyclo[3.2.1]octane moiety were isolated from the branches and leaves of I. merrillianum. The kind of neolignans with benzo-2,7-dioxabicyclo[3.2.1]octane bridged-ring was first encountered in nature. Moreover (+)−1 was found to have a better effect than racemate 1 in promoting NGF-induced neurite outgrowth of PC12 cells possibly by increasing the phosphorylation level of TrkA receptor, and MEK, ERK1/2 in Ras/ERK signaling cascade at concentrations of 1–10 μM, while its enantiomer (−)−1 played the opposite role at the same concentrations. Opposite absolute configuration leads to big difference between the interaction of compounds and the PC12 cells. However the phosphorylation level of Akt1 and Akt2 in the PI3K/Akt pathway showed no significant differences between (+)−1 and (−)−1. The results reported in this paper offer a pair of interesting neolignan enantiomers for potential NGF agonist and antagonist drug development.In conclusion, two neolignan enantiomers (+)−2, and MS3 were measured on the Agilent LC/MSD Trap XCT spectrometer and HRESIMS were performed on an Agilent 6520 Accurate-MS Q-TOF LC/MS system; Optical rotations were obtained with an Autopol VI ; CD spectra were recorded on a Chirascan spectrometer ; IR spectrum was acquired with a Bruker Vector-22 spectrometer with KBr pellets; Colum chromatography was performed on silica gel ; Reversed phase medium pressure liquid chromatography (RP-MPLC) was performed on a Buchi Sepacore system; TLC analysis was run on HSGF254 silica gel plates ; Preparative TLC was conducted with pre-coated silica gel HSGF254 plate .1D and 2D NMR spectra were recorded on a Bruker Ascend 500 NMR spectrometer with TMS as internal standard; ESIMS, MSI. merrillianum were collected in Gongshan county, Yunnan province, P. R. China, in August 2011, and authenticated by Prof. Han-Ming Zhang of Second Military Medical University. A voucher specimen (No. 20110815) is deposited in the School of Pharmacy, Second Military Medical University.The branches and leaves of I. merrillianum (20 kg) were extracted with 95% EtOH (3 × 80 L) for three times (3 × 1 hr) to afford a crude extract (1.7 kg) after removal of solvent under low pressure. The extract was suspended in water and partitioned with petroleum ether (488 g), EtOAc (836 g), and n-BuOH (526 g) successively. The petroleum ether extract was subjected to silica gel column chromatography (CC) , using petroleum ether-EtOAc gradient elution (50:1–0:1) to afford seven fractions A (51 g), B (86 g), C (95 g), D (16 g), E (21 g), F (129 g), G (17 g). Fraction F was subjected to RP-MPLC  to give five fractions F-1 (30 g), F-2 (21 g), F-3 (15 g), F-4 (31 g), F-5 (13 g). Fraction F-5 was further subjected to RP-MPLC  to give five fractions . Compound 1 (14.8 mg) was isolated from fraction F-53 by applying preparative TLC using petroleum ether-Me2O  developer.The air-dried and chopped branches and leaves of 1 was performed on a Chiralpak IA column (USA): 0.46 cm I.D. × 25 cm; Flow rate: 1.0 mL/min; Solvent: Hexane/DCM/DEA = 45/55/0.1 (v/v/v); Temperature: 30 °C; Wavelength: 254 nm; Injection volume: 10 μL for each sample; The retention time of (+)−1 (4.0 mg) and (−)−1 (5.0 mg) was 7.935 and 8.635 min, respectively, and the peak area ratio of (+)−1 to (−)−1 was 0.8:1 , [α]25 D of (−)−1: −48.0 ; CD of (+)−1  nm (Δε) 197 (−3.5), 210 (+5.8), CD of (−)−1  nm (Δε) 197 (+3.5), 210 (−5.8); IR (KBr) νmax 2935, 1618, 1508, 1465, 1204, 1034, 884 cm−1; HRESIMS m/z 417.1915 [M + H]+ ; 1H-NMR  δ: 1.36 , 1.65 , 3.03 , 3.55 , 3.74 , 3.77 , 3.82 , 3.83 , 3.85 , 4.49 , 6.39 , 6.45 , 6.46 , 6.86 ; 13C-NMR  δ: 115.2 (C-1), 147.9 (C-2), 96.8 (C-3), 152.3 (C-4), 142.5 (C-5), 112.6 (C-6), 38.9 (C-7), 106.0 (C-8), 22.6 (C-9), 117.9 (C-1′), 146.5 (C-2′), 100.3 (C-3′), 148.9 (C-4′), 143.1 (C-5′), 110.9 (C-6′), 47.9 (C-7′), 85.2 (C-8′), 20.7 (C-9′), 56.1 (OCH3-2), 56.2 (OCH3-4), 56.0 , 56.4 (OCH3-5′).colorless needle crystal (MeOH); mp. 58 ∼ 59 °C; [α]25 D of (+)−1 and (−)−1 were performed on an IBM cluster machine using Gaussian03 package. Conformational analysis for 7R,8S,7′S,8′S-1 and 7S,8R,7′R,8′R-1 was initially carried out via the Molecular Operating Environment (MOE) software package using the MMFF94 force field. The obtained conformers were further optimized, and verified as the true minima of potential energy surface using TDDFT at B3LYP/3-21G(d) basis set level. Conductor-like polarizable continuum model (CPCM) was adopted to consider solvent effects using the dielectric constant of methanol (ε = 32.6). The 21 lowest electronic transitions were calculated and the rotational strengths of each electronic excitation were given using both dipole length and dipole velocity representations. ECD spectra were stimulated using a Gaussian function with a half-bandwidth of 0.3 eV. Equilibrium populations of conformers at 298.15 K were calculated from their relative free energies (∆G) using Boltzmann statistics. The overall ECD spectra of (+)−1 and (−)−1 were then generated according to Boltzmann weighting of each conformers.The theoretical calculations of (+)−Conformational analysis was initially carried out using Maestro7.5 conformational searching, together with the MMFF94 molecular mechanics methods. MMFF94 structures were reoptimized using ab initio DFT at the B3LYP/3-21G (d) level. Energy of stable conformations were calculated and led to the relative energy, which in turn allowed the room temperature equilibrium populations to be calculated according to Maxwell - Boltzmann distribution law (Eqn 1).vel and length form Rlen in 10–40 erg-esu-cm/Gauss) between different states were calculated by time dependant density functional theory (TDDFT) at B3LYP/3-21G (d)//B3LYP/3-21G (d) level in methanol solution. All calculations are performed by the Gaussian03 program package.Excitation energy (in nm) and rotatory strength R  were obtained from the Cell Bank of Shanghai Institute of Biochemistry & Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences; NGF was purchased from Wuhan Hiteck Biological Pharma Co., Ltd. China; Dimethylsulfoxide (DMSO) and  (MTT) were purchased from Sigma ; Dulbecco’s Modified Eagle’s Medium (DMEM), a penicillin/streptomycin mixture, and fetal calf serum (FCS) and horse serum (HS) were purchased from Gibco ; Antibodies for p-TrkA, p-MEK, p-ERK1/2, p-Akt1 (Ser473), p-Akt2 (Thr308) and ERK1/2, Akt were obtained from Cell Signaling Technology ; Anti-mouse and anti-rabbit secondary antibody were purchased from Odyssey ; GAPDH  was purchased from Beyotime Biotechnology .2 at 37 °C. The tested compounds were dissolved in DMSO, and the final volume for DMSO in each well was no more than 0.1%. To rule out a potential influence of the solvent, we conducted a corresponding control experiments. PC12 cells were seeded in 96-well plate at a density of 5 × 104/well. After cell attachment overnight, each well was treated with different concentrations of test compounds or 0.1% DMSO for 24 h. Then 10 μL of MTT solution (5 mg/mL) was added to each well and the cultures were incubated for another 4 h at 37 °C. The supernatant was then removed and 100 μL of DMSO was added to each well and agitated at 60 rpm for 5 min to dissolve the precipitate. The absorbance was read at 570 nm by a SYNERGY microplate reader .The assay was performed in triplicate. PC12 cells were maintained in DMEM containing 5% FCS and 10% HS supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified atmosphere containing 5% CO4 cells/well in the above medium in 96-well plate. After cell attachment overnight each well was treated with compounds with or without 20 ng/mL NGF respectivly . After 24-h incubation, neurite outgrowth of the cells were determined by light microscopy . Neurite processes with a length equal to or greater than the diameter of the neuron cell body were scored as positive. Neurite outgrowth was determined from at least three different regions of interest in three independent experiments. Cell differentiation rate was calculated as the positive number of cells/total cell number. Data were expressed as the mean ± standard deviation (SD).Undifferentiated PC12 cells were plated in a density of 5 × 106 cells/well in six well palte. Then they were incubated with 20 ng/mL NGF and different concentrations of compounds for 30 min. After removal of the medium, PC12 cells were washed with ice-cold PBS, resuspended in lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 8.0), 0.02% NaN3, 0.01% PMSF, 0.2% Aprotinin, 1% TritonX-100 supplemented with protease inhibitor cocktail , and then centrifuged at 1,2000 rmp for 15 min. The concentration of total proteins was determined using a BCA kit . After boiling for 5 min the protein samples (30 μg) were electrophoresed on 10% SDS polyacrylamide gels and then transferred to polyvinylidene difluoride (PVDF) membrane. Nonspecific reactivity was blocked by 5% nonfat milk prepared in TBST  at room temperature for 1 h. The membranes were incubated with antibodies according to the manufacturers’ instructions. The image was captured by the Odyssey infrared imaging system . Protein densitometry was done using Quantity One imaging software (Bio-Rad) and normalized against GAPDH.PC12 cells were incubated at 37 °C for 24 h in a density of 1 × 10t-test, and multiple comparisons were compared by one-way ANOVA analysis of variance followed by Tukey post hoc test. Statistical significance was determined as P < 0.05.Data were analyzed using GraphPad Prism software . The comparison between two groups was analyzed by unpaired Student How to cite this article: Tian, X. et al. Distinctive effect on nerve growth factor-induced PC12 cell neurite outgrowth by two unique neolignan enantiomers from Illicium merrillianum. Sci. Rep.5, 16982; doi: 10.1038/srep16982 (2015).


The molecule is essentially planar (r.m.s. deviation = 0.028 Å). The molecules are linked by intermolecular N—H⋯O hydrogen-bonding interactions between the carbonyl and amine groups, forming a zigzag chain along the b-axis direction lying in a plane parallel to (-102). The chains are stacked along the c axis by π–π interactions [centroid–centroid distances = 3.6240 (3) and 3.5855 (4) Å]. A strong intramolecular N—H⋯O hydrogen-bonding interaction is observed between the nitro group and the amine group [2.660 (2) Å].Yellow crystals of the title compound 3-amino-4-nitrobenzyl acetate, C The chains stack along the c axis by π–π interactions [centroid–centroid distances = 3.6240 (3) Å  and 3.5855 (4) Å ].The crystal structure of 3-amino-4-nitrobenzyl acetate has interesting supramolecular features. The molecules are arranged in layers held together by intermolecular N2—H2on Fig. 2 lying inis Fig. 2 providesis Fig. 3 shows foet al. methanol: (5-amino-2-nitrophenyl)methanol was prepared by a modified literature protocol  was added dropwise by dropping funnel over 30 minutes. The reaction was stirred overnight at room temperature. The reaction was quenched with aqueous potassium hydroxide (2.45 M) until pH 11 was reached and continued to be stirred for 6 h, resulting in a greenish-brown solution. The solution was treated with a saturated solution of potassium carbonate followed by treatment with hydrochloric acid until pH 1 was reached. The reaction mixture was extracted with diethyl ether three times; organic portions were collected and dried with anhydrous sodium sulfate overnight. The solution was filtered under vacuum, the filtrate was collected and all solvent removed under rotary evaporation to give a green powder . 1H NMR,  δ: 4.61 , 4.95  , 6.03 , 6.63 , 7.07 , 8.02  methanol  and triethylamine  were dissolved in acetonitrile-d6 (0.7 mL) and added to an NMR tube. Acetic anhydride  was added to the tube via a syringe. The tube was held at room temperature overnight. On completion of the reaction the solvent was removed in vacuo and the residue was reconstituted in a minimum amount of methylene chloride. The sample was loaded on a column of silica and eluted with an ethyl acetate/hexane solution (70/30 v/v %). The separated solutions were allowed to slowly evaporate at room temperature. The parent compound (5-amino-2-nitrobenzyl acetate) elutes first and is isolated as a yellow powder. 1H NMR  δ: 2.10 , 4.35 , 5.50 , 6.55 , 6.68 , 8.09   δ: 2.19 , 5.53 , 7.44 , 7.65 , 7.75 , 8.15 .Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015008750/pk2548sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989015008750/pk2548Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015008750/pk2548Isup3.cmlSupporting information file. DOI: 1063364CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive T cell-mediated immunity, the ILC2 and T cell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced T cell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity.Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s in vivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific T cells to instigate a dialog in which IL-2 production from T cells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing   •Genetic ablation of ILC2s impairs type-2 immunity•MHCII-expressing ILC2s potentiate Th2 responses•IL-2 from T cells promotes ILC2 proliferation and expression of type-2 cytokines•N. brasiliensis expulsionMHCII and IL-13 expression by ILC2s is important for  Type-2 innate lymphoid cells proliferate and release interleukin-13 during protective immunity to helminth infection and detrimentally during allergy and asthma. McKenzie and colleagues establish that these activities are potentiated through an MHC class II-mediated dialogue with T cells. Group 2 immunity is believed to have evolved to combat parasitic helminth infection, but also contributes to wound healing. These responses are characterized by adaptive T helper 2 (Th2) cells expressing interleukin-4 (IL-4), IL-5, and IL-13, B cells secreting immunoglobulin E (IgE), eosinophils, and mast cells. Innate lymphoid cells (ILCs) also play key roles in type-2 responses by producing high amounts of type-2 cytokines . ILC2s aPrevious studies have demonstrated that MHCII-expressing conventional antigen-presenting DCs play a critical role in generating type-2 responses . MHCII iNippostrongylus brasiliensis.MHCII is expressed on ILC2s and ILC3s . MHCII oN. brasiliensis infection. Concurrently, the impaired Th2 cell responses that were observed in these mice implied a role for ILC2s in promoting adaptive immunity  enabling the CD4-cre-mediated excision of the DTR gene from T cells but its retention in ILC2s -cre (restricted to the lymphoid lineage) mice yields an ILC2-deficient mouse strain in which other lineages are unaffected  is critical for ILC2 development . We haveaffected A–S2C. Rocontrols F, this scontrols D. The Roed ILC2s G and a d T cells H.+ Th2 cells. These data support an in vivo interplay between ILC2s and CD4+ T cells, and we proceeded to investigate the potential role of ILC2-expressed MHCII in this interaction.Thus, in two distinct ILC2-deficient mouse strains in which ILC2 ablation is accomplished through discrete pathways, we observed concordant in vivo defects in CD4–Klrg1+CD25+ST2+ICOS+Sca1hiGATA3+IL-13+) and those elicited in response to IL-25 or IL-33, as compared to ILC2s from MHCII-deficient mice  (+ T cells via this molecule.We confirmed MHCII expression on ∼50%–70% of naive wild-type (WT) ILC2s (LinhcII−/−) A–S3C. ExhcII−/−) C. Lymph hcII−/−) D and S3EhcII−/−) D, which + cells  are impN. brasiliensis-infected IL-13-deficient mice. The transferred ILC2s could be identified in the IL-13-deficient recipients due to expression of IL-13 protein -specific OTII transgenic (Tg) C57Bl/6 CD4 T cells  in the p T cells A. In conmination B and lim T cells C. An irr T cells D. ILC2s  T cells E also fa T cells E. The in T cells E. The petoid DCs B.Figure + T cells  (ILC2s endocytosed soluble antigen in vitro dose dependently G–S5I andb) . ILC2s wb) I. Thus, + T cells toward a Th2 phenotype. Supernatants from OTIITg T cells cocultured with OVA peptide-pulsed ILC2s contained markedly increased amounts of type-2 cytokines IL-5, IL-6, IL-9, and IL-13, but little IFN-γ or IL-17A, as compared to controls lacking peptide  . Cocultu+ T cell interaction. Notably, although IL-4, a factor linked to Th2 cell polarization, is not always detectable in ILC2s  is highly expressed on ILC2s. Indeed, culture of ILC2s with IL-2 resulted in increased numbers of IL-13-producing ILC2s C. IntracRag2−/− mice have impaired worm clearance. To determine whether this deficit might be due to insufficient IL-2 production from T cells failing to induce sufficient ILC2s, we infected Rag2−/− mice with N. brasiliensis while also treating one group with an IL-2-anti-IL-2 antibody complex to facilitate slow release of the active IL-2 molecule. Rag2−/− mice sufficient for ILC2s failed to expel their worms efficiently  receptor for their development and proliferation, and are absent in γc receptor-deficient mice . FurtherN. brasiliensis-infected MHCII-sufficient mice, MHCII trogocytosis was insufficient to restore worm expulsion. In contrast to mouse ILC2s, we found that human ILC2s process whole Der p 1 antigen and present the derived peptides to T cells in vitro. Thus, our existing in vitro cellular proliferation assays appear too insensitive. Previously, human NK cells have been shown to process and present tetanus toxoid and Der p 1 to T cells, but protein antigen from Mycobacterium leprae was poorly processed and presented  (Il13egfp/egfp (Il7rCre (Rorasg/+ (JAX Laboratories) mice were on a C57Bl/6 background. Il13tdTomato/+, Il4gfp/gfp (Il4−/−Il5−/−Il9−/−Il13−/− (QUAD-KO)  , Il13egfgfp/egfp , Il7rCre  mice werN. brasiliensis infection. DTx  was administered daily for up to 5 days i.p. Mice were euthanized 24 hr later and tissues collected for analysis. Control animals received PBS only.IL-25 (either 2 μg/mouse or 0.5 μg/mouse of recombinant mouse IL-25 [Centocor] in PBS) was administered daily for 3 days intraperitoneally . For i.p. IL-33 administration, 0.5 μg/mouse or 1 μg/mouse recombinant mouse IL-33 (BioLegend) in PBS was administered daily for up to 4 days (termed IL-33-elicited ILC2s). For intranasal IL-33 administrations, mice were anesthetized and 0.4 μg of recombinant protein administered in PBS on 4 consecutive days. IL-2 complexes were prepared by incubating IL-2 and anti-IL-2 mAb (clone JES6) (both from R&D Systems) at a 1:5 w/w ratio for 20 min at 37°C. Complexes were then diluted in PBS and administered i.p. (1 μg IL-2 and 5 μg anti-IL-2 per dose) on days 0, 2, 4, 6, and 8 following + B6/SJL mice were reconstituted with a 1:1 mixture of bone-marrow cells (2 × 106 cells) containing either CD45.1+ B6/SJL and CD45.2+ C57Bl/6 or CD45.1+ B6/SJL and CD45.2+MhcII−/− cells. After 10 weeks, mice were given daily doses of recombinant mouse IL-33 (0.4 μg per dose).For mixed-chimera experiments, lethally irradiated CD45.1−  ICOS+. For IL-7/IL-33 in vitro cultures of ILC2s, we followed the protocol as described previously (4 cells per well in the presence or absence of IL-2 (10 ng/ml). Cultures were maintained for 3 days at 37°C.For the expansion of ILC2s in vitro from WT mice, ILC2s were FACS purified, as defined by LINeviously . CultureMhcII−/−, or QUAD-KO mice, were resupended in RPMI 1640 (GIBCO®) supplemented with 2 mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, and 10% Hyclone Fetal Bovine Serum (Thermoscientific). Where necessary, ILC2s were pulsed with OVA-peptide (323–339) for 2 hr at 37°C. Subsequently ILC2s were washed thoroughly and 1 × 104 ILC2s were cultured with 1 × 104 labeled CD3+CD4+ OTIITg or DO11.10Tg splenic T cells, which were sorted by flow cytometry, for 5 days at 37°C, 5% CO2. Where indicated, the antibodies M5/114.15.2 , 16-10A1 , and GL-1  were added to cultures at a concentration of 1 μg/ml. 11B11  was incubated at a concentration of 10 μg/ml. Where appropriate, blockade of IL-2 was performed with 10 μg/ml of JES6-1A12 (eBioscience) and S4B6 (BD PharMingen). Cocultures containing in vivo-loaded OVA-DQ+ ILC2s were prepared by FACS-purifying ILC2s, based on Lin−ICOS+OVA-DQ+ or lineage−ICOS+OVA-DQ−, from the bronchoalveolar lavage of WT mice receiving an intranasal administration of OVA-DQ and IL-33 on 7 consecutive days. Cocultures were setup at a 1:1 ratio with 4 × 104 cells per well. Human samples were taken under GCP guidance with ethical approval of the NRES Committee South Central.Freshly isolated or in vitro cultured ILC2s, from WT, N. brasiliensis larvae. Where appropriate, FACS-purified ILC2s were transferred by intravenous injection 2 hr after N. brasiliensis injections.Mice were inoculated subcutaneously with 500 viable third-stage b) prior to flow cytometric analysis.To assess the ability of ILC2s to present processed antigen, we cultured ILC2s in the presence of 100 μg/ml E-alpha (Eα)-green fluorescent protein (GFP) fusion protein for 20 hr. An Eα-specific antibody (clone YAe) was used to detect an Eα-derived peptide (Eα52-68) bound in the context of MHC (I-AP < 0.05 considered significant.Graph Pad Prism was used to calculate the SEM when different numbers of data sets existed in each experimental group. When data were normally distributed and when two independent variables were being analyzed, a Kruskal-Wallis one-way ANOVA with Bonferroni post-analysis was performed. In all other instances, statistical differences between groups were calculated with Student’s t test, with A.N.J.M., S.H.W, Y.Y.H., and C.J.O conceived the study. C.J.O, S.H.W., J.A.W., J.L.B., Y.Y.H., E.H., A.E., S.T.S., and P.G.F. did the experiments or contributed to experimental design, reagents, and analysis. G.S.O and M.S. performed the human ILC2 experiments. A.N.J.M. wrote the manuscript with contributions from all authors, but primarily from C.J.O and J.A.W.


I atom with the unsymmetrical ligand N-(pyridin-4-ylmethyl)pyridine-3-amine afforded a helical chain. The AgI atom adopts a slightly distorted linear coordination geometry. The symmetry-related right- and left-handed helical chains are alternately arranged via Ag⋯Ag interactions and π–π stacking interactions, resulting in the formation of a two-dimensional supramolecular network.The reaction of Ag 11H11N3)]NO3·2(CH3)2SO}n, comprises one AgI atom, one N-(pyridine-4-ylmethyl)pyridine-3-amine ligand, one nitrate anion and two dimethyl sulfoxide molecules. The AgI atoms are bridged by two pyridine N atoms from two symmetry-related ligands, forming a helical chain and adopting a slightly distorted linear coordination geometry [N—Ag—N = 175.37 (8)°]. The helical chain, with a pitch length of 16.7871 (8) Å, propagates along the b-axis direction. In the crystal, symmetry-related right- and left-handed helical chains are alternately arranged via Ag⋯Ag interactions [3.4145 (4) Å] and π–π stacking interactions [centroid–centroid distance = 3.650 (2) Å], resulting in the formation of a two-dimensional supramolecular network extending parallel to (100). Weak Ag⋯O  interactions, as well as several N—H⋯O and C—H⋯O hydrogen-bonding interactions, contribute to the stabilization of the crystal structure. Parts of the dimethyl sulfoxide solvent molecule are disordered over two sets of sites in a 0.937 (3):0.063 (3) ratio.The asymmetric unit of the title compound, {[Ag(C I cation, but also their potential applications as functional materials pyridine-3-amine, which was been synthesized by the reaction of 3-aminopyridine and pyridine-4-carboxaldehyde according to literature methods pyridine-3-amine ligand, one nitrate anion and two DMSO molecules. The S atom of one of the DMSO molecules is disordered over two sites [site-occupancy factors of 0.937 (3) for S2 and 0.063 (3) for S2′]. The Ag atom links two pyridine N atoms from two symmetry-related ligands, forming a helical chain. Thus the AgI atom is two-coordinate in a slightly distorted linear coordination geometry [N—Ag—N = 175.37 (8)°], with the Ag—N bond lengths of 2.158 (2) and 2.162 (2) Å. The helical chain propagates along the b-axis direction pyridine-3-amine ligand, the two pyridine rings are almost perpendicular, the dihedral angle between their mean planes being 86.28 (7)°.The molecular components of the title structure are shown in Fig. 1on Fig. 2 and its  al. 2013. The twovia Ag⋯Ag interactions [3.4145 (4) Å], resulting in the formation of a two-dimensional supramolecular network extending parallel to (100)  and Ag1⋯O4 = 2.690 (2) Å; symmetry code: (i) −x + 1, −y + 1, −z]  Fig. 2. π–π staz] Fig. 2, as wellz] Fig. 2. In addiet al. (2013The structures of the silver(I) nitrate and perchlorate complexes of the same ligand have been reported as their monohydrated and non-solvated forms, respectively, by Zhang  al. 2013. These cN-(Pyridin-4-ylmethyl)pyridine-3-amine was prepared according to the procedure described by Lee et al.  = 1.2Ueq.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S1600536814024817/hg5419sup1.cifCrystal structure: contains datablock(s) I, New_Global_Publ_Block. DOI: 10.1107/S1600536814024817/hg5419Isup2.hklStructure factors: contains datablock(s) I. DOI: 1033712CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title compound, 5-[4-(diethylamino)phenylmethylidene]-2,2-dimethyl-1,3-dioxane-4,6-dione, have been synthesized and its crystal structure determined. Due to the absence of hydrogen-bond donors in the structure, the crystal packing is controlled by van der Waals forces and weak C—H⋯O interactions, which associate the molecules in dimers. 17H21NO4, consists of substituted Meldrum’s acid with a [4-(diethylamino)phenyl]methylidene fragment attached to the fifth position. The heterocycle assumes a distorted boat conformation. The planar part of heterocycle is almost coplanar with the benzene ring due to the presence of a long conjugated system in the molecule. This leads to the formation of C—H⋯O-type intramolecular contacts. As a result of the absence of hydrogen-bond donors in the structure, the crystal packing is controlled by van der Waals forces and weak C—H⋯O interactions, which associate the molecules into inversion dimers.The title compound, C Trost & Maruniak, 2013e.g. Mohite & Bhat, 2013et al.  are attractive building blocks in organic chemistry: these compounds are used for the synthesis of different heterocycles. Recent examples include: pyrazolidinones phenyl]methylidene fragment attached to fifth position  Å.π–π stacking interactions are also observed between conjugated systems of the molecules. The distance between the corresponding least-square planes is 3.54 et al., 2015The crystal structure of the zwitterionic form of 5-[4-(diethylamino)benzyl]-2,2-dimethyl-1,3-dioxane-4,6-dione has been already reported phenylmethylidene]-2,2-dimethyl-1,3-dioxane-4,6-dione was obtained from Meldrum’s acid  and 4-diethylaminobenzaldehyde  by heating in water (50 ml) at 348 K for 2 h, followed by cooling to room temperature and filtration of the formed precipitate and recrystallization from ethanol  analogously to the method described previously (Mierina Uiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015017673/xu5872sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989015017673/xu5872Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015017673/xu5872Isup3.cmlSupporting information file. DOI: 1426237CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal of (1), molecules are linked by C—H⋯O hydrogen bonds forming chains along [100], while in the crystal of (2), molecules are linked by C—H⋯O and C—H⋯Cl hydrogen bonds forming slabs parallel to (001).In the related title compounds, (1) and (2), the 3-thiazolidine ring pucker is twisted on the S—C 15H11Cl2NOS, (1), and C15H11Cl2NO2S, (2), wherein (2) is the oxidized form of (1), the thiazolidine ring is attached to two chlorophenyl rings. The chlorophenyl ring on the 2-carbon atom position points in the same direction as that of the S atom in (1), while in (2), the S atom points in the opposite direction. The O atom on the chiral S atom in (2) is trans to the chlorophenyl ring on the 2-carbon. The chlorophenyl ring planes in each structure are close to orthogonal, making dihedral angles of 78.61 (6) and 87.46 (8)° in (1) and (2), respectively. The thiazolidine ring has a twisted conformation on the S—Cmethine bond in (1), and an envelope conformation with the S atom 0.715 (3) Å out of the plane of other four atoms in (2). In the crystal of (1), molecules are linked by C—H⋯O hydrogen bonds, as well as by slipped parallel π–π interactions [inter-centroid distance = 3.840 (3) Å] between inversion-related phenyl rings, forming sheets parallel to (001). In the crystal of (2), molecules are linked via C—H⋯O and C—H⋯Cl hydrogen bonds, forming slabs parallel to (001).In the crystal structures of the title compounds, C Steric strain was further relieved by twisting so that both the aryl ring on C1 and the oxygen on S1 became pseudo-axial.Comparison of the two structures shows that the oxygen–sulfur bond in (2) formed on the less hindered side of compound (1), away from the aryl group on C1, leading to a via C—H⋯O hydrogen bonds, forming chains along [100]; see Table 1via slipped parallel π–π interactions involving inversion-related chlorophenyl rings, leading to the formation of sheets parallel to (001) .In the crystal of (1), molecules are linked via by C—H⋯O and C—H⋯Cl hydrogen bonds, forming slabs parallel to (001); see Table 2In the crystal of (2), molecules are linked et al., 2014p-chlorine atoms on both phenyl rings, and the compound does not have a twist in the thiazine ring. Compound (2) is related to 2-aryl-1,3-thiazolidin-4-one 1-oxides, viz. 3-butyl-2-phenyl-1,3-thiazolidine-1,4-dione -3, 5-dimethyl-1-oxo-2-phenyl-4-thiazolidinone thiazolidin-4-one 1-oxide  differs from the previously reported 2,3-diphenyl-1, 3-thiazolidin-4-one (Yennawar Compound (1): prepared as previously reported : 2,3-bis (4-chlorophenyl)-1,3-thiazolidin-4-one (1)  was added to a 25 ml round-bottom flask. Methanol (4 ml) was added and the mixture was stirred at room temperature before cooling to 273–278 K. A solution of Oxone  in distilled water (4 ml) was prepared. This solution  was slowly added to the reaction mixture with stirring at 273–278 K. The reaction was followed by TLC. An additional aliquot of Oxone solution (0.67 ml) was added to convert the remaining starting material to sulfoxide. The mixture was extracted three times with methylene chloride. The organic layers were combined and washed with water and saturated NaCl, then dried over sodium sulfate. The solution was concentrated under vacuum to give compound (2) as a crude solid. The solid was recrystallized from a mixture of methylene chloride and hexane, and then dried . Colourless plate-like crystals were obtained by slow evaporation of a solution in ethanol.Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details for structures (1) and (2) are summarized in Table 310.1107/S2056989015001954/su5062sup1.cifCrystal structure: contains datablock(s) 1, 2, global. DOI: 10.1107/S2056989015001954/su50621sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989015001954/su50622sup3.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989015001954/su50621sup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015001954/su50622sup5.cmlSupporting information file. DOI: 1046346, 1046345CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: