DDAA hydrogen-bonded array. Hirshfeld surface analysis was employed to further examine the intermolecular interactions.The asymmetric unit consists of a 2,4-diamino-6-phenyl-1,3,5-triazin-1-ium cation and a 4-methylbenzoate anion. The protonated nitrogen and amino group nitrogen atoms are involved in hydrogen bonding with the sulfonate oxygen atoms through a pair of intermolecular N—H⋯O hydrogen bonds. The inversion-related molecules are further linked by four N—H⋯O intermolecular interations to produce a complementary  9H10N5+·C7H7O3S−, the asymmetric unit consists of a 2,4-diamino-6-phenyl-1,3,5-triazin-1-ium cation and a 4-methylbenzenesulfonate anion. The cation is protonated at the N atom lying between the amine and phenyl substituents. The protonated N and amino-group N atoms are involved in hydrogen bonding with the sulfonate O atoms through a pair of intermolecular N—H⋯O hydrogen bonds, giving rise to a hydrogen-bonded cyclic motif with R22(8) graph-set notation. The inversion-related molecules are further linked by four N—H⋯O intermolecular interactions to produce a complementary DDAA  hydrogen-bonded array, forming R22(8), R42(8) and R22(8) ring motifs. The centrosymmetrically paired cations form R22(8) ring motifs through base-pairing via N—H⋯N hydrogen bonds. In addition, another R33(10) motif is formed between centrosymetrically paired cations and a sulfonate anion via N—H⋯O hydrogen bonds. The crystal structure also features weak S=O⋯π and π–π interactions. Hirshfeld surface and fingerprint plots were employed in order to further study the intermolecular interactions.In the title molecular salt, C Here thly Fig. 2. The invCg1; Y—X, π = 46.33°],  is based on the distances from the nearest atom inside (di) and outside (de) the surface. The three-dimensional dnorm surface of the title compound is shown in Fig. 4dnorm values on the surface corresponding to N—H⋯O and N—H⋯N interactions. Two-dimensional fingerprint plots are shown in Fig. 5Hirshfeld surface analysis  of 2,4-diamino-6-phenyl-1,3,5-triazine (0.187 g) and a hot methanolic solution (10 ml) of 4-methylbenzene sulfonic acid (0.172 g) in 1:1 molar ratio. The reaction mixture was warmed over a water bath for a few minutes. The resultant solution was then allowed to cool slowly at room temperature. After a few days, colourless block-shaped crystals were separated out.Uiso(H) set to 1.2–1.5Ueq(C) or 1.3Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018010368/jj2200sup1.cifCrystal structure: contains datablock(s) global, I. DOI: Click here for additional data file.10.1107/S2056989018010368/jj2200Isup2.cmlSupporting information file. DOI: 1820866CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


This data is available at the Metabolomics Workbench,  Specime22.1Tcf/LacZ mouse is a transgenic canonical Wnt reporter line that allows localization of active Wnt/β-catenin signaling LacZ transgene leads to induction of LacZ/β-galactosidase  expression wherever Wnt signaling is active. The Tcf/LacZ strain was previously backcrossed onto the C3H background All procedures involving mice were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care and Use Committee at the University of Miami. The optic nerve crush and associated treatments were done on Tcf/LacZ mice. As described previously Optic nerve crush (ONC) injury was performed as described previously Regenerating RGC axons were anterogradely labeled using cholera toxin β subunit (CTB) with a conjugated Alexafluor 546  that was intravitreously injected in a volume of 2 μL at two days prior to animal euthanasia. The animals were then perfused using 4% paraformaldehyde and eyes and optic nerves were dissected and processed through a 5–20% sucrose gradient and then embedded in OCT Compound (Tissue Tek) for cyrosectioning. Longitudinal optic nerve sections were cut at a thickness of 8 μm and mounted onto slides and then imaged using a fluorescent microscope (Zeiss).2.22.2.14 mL of methanol (LC-MS grade) and 2 mL of chloroform (LC-MS grade) were added to each sample. After 2 min vigorous vortexing and 2 min sonication in ultrasonic bath, the samples were incubated at 48 °C overnight . The following day, 2 mL of water (LC-MS grade) and 1 mL of chloroform were added, samples vigorously vortexed for 2 min and centrifuged at 3000 RCF, 4 °C for 15 min to obtain phase separation. Lower phases were collected and dried in a centrifugal vacuum concentrator. Samples were stored at −20 °C until reconstituted in 150 μL of chloroform:methanol (1:1) prior to mass spectrometric analysis. Samples list is in 2.2.2Reversed phase chromatographic separation utilized Accela Autosampler (Thermo), Accela 600 pump (Thermo) and Acclaim C30 column: 3 μm, 2.1 × 150 mm (Thermo). The column temperature was maintained at 30 °C and tray temperature at 20 °C. Solvent A was composed of 10 mM ammonium acetate (LC-MS grade) in 60:40 methanol:water (LC-MS grade) with 0.2% formic acid . Solvent B was composed of 10 mM ammonium acetate with 60:40 methanol:chloroform with 0.2% FA. The flow rate was 260 μL/min and injection volume was 10 μL. The gradient was 35–100% solvent B over 13.0 min, 100% solvent B over 13.0–13.8 min, 100-35% solvent B over 13.8–14.5 min, 35% solvent B over 14.5–18.0 min, 0% solvent B over 18.0–20.0 min.2.2.36 ions and maximum ion injection time (IT) of 100 ms. Data-dependent MS/MS (top10) were acquired using the following parameters: resolution 17,500; AGC 1 × 105; maximum IT 75 ms; 1.3 m/z isolation window; underfill ratio 0.1%; intensity threshold 1 × 103; dynamic exclusion time 3 s. Normalized collision energy (NCE) settings were: 15, 30, 45, 60, 75, 90 . Total instrument time ∼216 h. External mass calibration was performed using the standard calibration mixture. Each sample was preceded and followed by a blank run (1:1 chloroform:methanol).The Q Exactive (Thermo) mass spectrometer was operated under heated electrospray ionization (HESI) in positive and negative mode separately. The spray voltage was 4.4 kV, the heated capillary was held at 310 °C (negative mode) or 350 °C (positive mode) and heater at 275 °C (positive mode). The S-lens radio frequency (RF) level was 70. The sheath gas flow rate was 30 (negative mode) or 45 units (positive mode) and auxiliary gas was 14 (negative mode) or 15 units (positive mode). Full scan used resolution 70,000 with automatic gain control (AGC) target of 1 × 102.2.4Lipid identification and relative quantification were performed using LipidSearch 4.1 software (Thermo). The search criteria were as follows: product search; parent m/z tolerance 5 ppm; product m/z tolerance 5 ppm; quantification: m/z tolerance 5 ppm, retention time tolerance 1 min. The following adducts were allowed in positive mode: +H, +NH4, +H-H2O, +H-2H2O, +2H, +Na, +K and negative mode: -H, +HCOO, +CH3COO, -2H, -Cl. All classes were selected for search. LipidSearch nomenclature is used .Table 2L2.2.5Positive and negative mode identifications at different NCE were aligned in LipidSearch, allowing calculation of unassigned peaks. The following settings were applied: product search; alignment method max; retention time tolerance 0.1 min; filters: toprank, main isomer peak; M-score 5; molecular lipid identification grade: A-B (A: lipid class and fatty acid completely identified or B: lipid class and some fatty acid identified). Peaks of the following lipid classes: BisMePA, SQMG, MGDG and MGMG, were considered false positives and were removed. Only peaks appearing in all replicates were accepted. Peaks with the same annotated lipid species were merged. Lists of species and their main areas were uploaded to Metaboanalyst 4.0


The location of Vγ6+ γδ T cells, which peaked in the newborn thymus, was associated with mTEC. In vivo administration of clone 1C10-1F7 mAb impaired protection against Klebsiella pneumoniae infection but ameliorated psoriasis-like dermatitis induced by imiquimod treatment. These new mAbs are useful to elucidate the development, location, and functions of Vγ6 γδ T cells in mice.There are seven Vγ gene segments in the TCR γ chain loci of mice. We developed monoclonal antibodies (mAbs) specific to the Vγ6 chain (Heilig & Tonegawa nomenclature). By immunizing Vγ4/6 KO mice with complementarity-determining region peptides in Vγ6 chains, we generated three hybridomas. These hybridomas produced mAbs capable of cell surface staining of Vγ6/Vδ1 gene–transfected T-cell line lacking TCR as well as of Vγ1 At the periphery, most of the spleen and LN γδ T cells express Vγ1 and Vγ4, whereas Vγ7-expressing γδ T cells are more prevalent in intestinal intraepithelial cells (i-IELs)  protein of the B7 superfamily using germ line–encoded motifs distinct from CDRs within the Vγ7 chain  and one nonfunctional pseudo Cγ gene (φCγ3), four joining segments, including one pseudogene , and seven variable (Vγ) gene segments . The Vγ (i-IELs) . This biγ7 chain . Thus, tbright γδ T cells by anti-CD3ε mAb. However, detailed characteristics of Vγ6+ γδ T cells remain obscure because of the lack of Vγ6-specific mAb. In this study, we developed new mAbs specific to the murine Vγ6 chain and report the successful production, characterization, and in vitro effects of a novel anti-Vγ6 mAb with potential applications in elucidating roles of Vγ6 γδ T cells in infection and inflammation in mice.All monoclonal antibodies (mAbs) specific to Vγ chains, except for Vγ3 and Vγ6, are currently available for cell surface staining . We have21–35) and CDR2 (Vγ650–66) in Vγ6 chains (21–35) and CDR2 (Vγ650–66), respectively by ELISA.The V part of an Ab, including the unique Ag-binding site, is known as the idiotype. When one Ab binds to an idiotype of another Ab, it is referred to as an anti-idiotypic Ab . An anti6 chains . Vγ4/6 K6 chains . The sup50–66) peptide.To further select mAbs available for cell surface staining, we screened for those mAbs capable of staining TG40, a cell surface TCR–negative and intracytoplasmic CD3–positive mutant of the 21.2.2 mouse T-cell line , which w17D1 mAb, which detects dETC-bearing Vγ5/Vδ1 , reporteWe next determined the nucleotide sequences and the reduced aa sequences of the H and L chains of Vγ6-specific mAbs of 1C10-1F7, 2G2-2A3, and 5E10-C12. The V-D-J genes of the H chain, the V-J genes of the L chain of these mAbs, and aa sequences of each CDR3 are shown in + γδ T cells in the nonlymphoid tissues from C57BL/6, BALB/c, and Vγ4/6 KO mice with 1C10-1F7, 2G2-2A3, or 5E10-C12 mAbs. Most of the Vγ1− Vγ4− Vγ5− γδ T cells in the reproductive organs and large proportions of the Vγ1− Vγ4− Vγ5− γδ T cells in the PEC and c-LPL from C57BL/6 were positively stained with 1C10-1F7 mAb , lungs, and c-LPL as tissue-associated cells . We stai-1F7 mAb  and S1A.e organs . The Vγ6-1F7 mAb . The Vγ6 KO mice . We sele-1F7 mAb . dETCs, -1F7 mAb  and i-IE T cells , were no T cells . To furtiversity . This rel thymus , reprodul thymus , and thel thymus .+ γδ T cells by immunohistochemical analysis with 1C10-1F7 mAb in the uterine cervix, in which most of γδ T cells were positively stained with 1C10-1F7 mAb. Consistent with a previous report  resulted in failure to generate Vγ5s per se . We alsowere not . These r− CD8− double-negative (DN)2b stage, which is located in the medulla  gene, which is expressed by the mTEC  and substantially reduced number of mTECs in thymus  (high TCRδint γδ T cells were Vγ6+ γδ T cells and that IL-17A–producing CD3int TCRδhigh γδ T cells were Vγ4+ γδ T cells (in (BCG)  and stai T cells .+ γδ T cells. + γδ T cells became invisible in the PEC, reproductive organs, and lungs on day 3 after in vivo administration of 1C10-1F7 mAb , whereas γδ T cells with a low intensity of Vγ6 TCR recovered in these organs by day 6 after administration   . Upon IMmed skin . Howevermed skin . These r KO mice . Block o KO mice . However KO mice .+ γδ T cells. These mAbs are available for flow cytometry, immunohistochemistry, and in vivo function analysis. Vγ6+ γδ T cells play important roles in protection against microbial infection and in pathogenesis of inflammatory diseases such as colitis and autoimmune diseases by producing IL-17A  and CDR2 (Vγ650–66) in Vγ6 chains. Vγ4/6 KO mice were immunized with the KLH-conjugated peptides emulsified with CFA. These mice were reimmunized with the KLH-conjugated peptides without CFA 17 d after the first immunization. Iliac LNs were collected 21 d after the last immunization and were fused with SP2/0-Ag14 using polyethylene glycol.For immunization, two types of peptides were synthesized from truncated regions of CDR1  including 1 ng/ml recombinant human IL-6 (R&D systems). After 7–10 d of culture, culture supernatants were collected. For flow cytometry and immunohistochemical analysis, new anti-Vγ6 mAbs were purified from the hybridoma supernatant using the mouse TCS purification system (Abcam) and conjugated with Alexa Fluor 647 using a labeling kit (Invitrogen). For the 1C10-1F7 administration experiment, 1C10-1F7 mAb was purified from the hybridoma supernatant using the Protein G Spin kit (Thermo Fisher Scientific).Total RNA was isolated and purified from hybridomas with RNeasy Plus Universal Mini Kit (QIAGEN). Total RNA was converted to complementary DNA (cDNA) with Superscript III reverse transcriptase (Invitrogen). Next, BCR genes were amplified using adaptor ligation-mediated PCR . High-thg for 20 min in a 40% and 70% Percoll  gradient.Single-cell suspensions were isolated from the thymus, PEC, reproductive organs (vagina/uterine cervix), i-IEL, c-LPL, and lungs as previously described . EpidermCells were stained for 20 min at 4°C with mAbs. We added 1 μg/ml propidium iodide (Sigma-Aldrich) to the cell suspension just before flow cytometry to detect and exclude dead cells from the surface staining analysis. To measure cytokine production, the cells were stimulated with 25 ng/ml PMA (Sigma-Aldrich) and 1 μg/ml ionomycin (Sigma-Aldrich) for 5 h at 37°C; 10 μg/ml Brefeldin A (Sigma-Aldrich) was added for the last 4 h of incubation. After the cells were stained with various mAbs, intracellular staining was performed according to the manufacturer’s instructions (BD Biosciences). 100 μl BD Cytofix/Cytoperm solution (BD Biosciences) was added to the cell suspension with gentle mixing and incubated for 20 min at 4°C. Fixed cells were washed twice with 250 μl 10% BD Perm/Wash solution (BD Biosciences) and then stained intracellularly for 30 min at 4°C. Stained cells were analyzed on a FACSVerse flow cytometer (BD Biosciences) and data were analyzed using FlowJo software (Tree Star). Abs for flow cytometric analysis used in this study: PerCP-Cy5.5–conjugated anti-MHC class II (M5/114.15.2), APC-Cy7–conjugated anti-CD3ε (145-2C11), PE-Cy7–conjugated anti-CD45.2 (104), anti-Vγ4 (UC3-10A6) V500-conjugated anti-MHC class II (M5/114.15.2) mAbs, and streptavidin were purchased from BD Biosciences. PE-conjugated Hamster IgG isotype control (HTK888), PerCP-Cy5.5-conjugated anti-IL-17A (ebio17B7), biotin-conjugated anti-CD45.1 (A20), Purified Mouse IgG1 κ isotype control (P3), and Purified Mouse IgG2b κ isotype control (eBMG2b) mAbs were all purchased from eBioscience. FITC-conjugated, anti-MHC class II (M5/114.15.2), anti-Vγ1 (2.11), anti-Vγ4 (UC3-10A6), PE-conjugated anti-Vγ1 (2.11), anti-Vγ4 (UC3-10A6), anti-mouse IgG (Poly4053), APC-conjugated, anti-TCRδ (GL3), anti-Vγ4 (UC3-10A6), Alexa Fluor 647–conjugated anti-CD3ε (17A2), Mouse IgG1 κ isotype control (MOPC-21), anti-mouse IgG (Poly4053), PE-Cy7–conjugated anti-TCRδ (GL3), V421-conjugated anti-TCRδ (GL3), and biotin-conjugated anti-rat IgM (MRM-47) mAbs were purchased from BioLegend. PE-conjugated anti-Vγ5 (536) mAb was purchased from Santa Cruz Biotechnology. Anti-Vγ7 (F2.67) and 17D1 mAbs were collected from F2.67 and 17D1 hybridoma culture supernatant.+ γδ T cells were sorted using FACSAria (BD Bioscience). Total RNA was purified from sorted 1C10-1F7+ γδ T cells using an RNeasy Mini kit (QIAGEN), and cDNA was synthesized using Superscript II (Invitrogen) according to the manufacturer’s instructions. PCR was performed on a PCR thermal cycler (Takara Corp.). RT-PCR products were analyzed by blotting in 1.8% agarose gels. For RT-PCR analysis of Vγ6 TCR gene, combinations of following primers were used. Forward primers: Vγ6, 5′-GGAATTCAAAAGAAAACATTGTCT-3′. Reverse primers: Cγ, 5′-CTTATGGAGATTTGTTTCAGC-3′. Forward primers: β-actin, 5′-TGGAATCCTGTGGCATCCATGAAAC-3′. Reverse primers: β-actin, 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′. Purified Vγ6 PCR products of 1C10-1F7+ γδ T cells from PEC were sequenced using BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) and 3500xL Genetic Analyzers (Applied Biosystems).Single-cell suspensions were isolated from PEC and stained with mAbs. 1C10-1F7Uterine cervixes from 8-wk-old WT mice and thymus from 0-d-old WT mice were fixed with phosphate-buffered 4% paraformaldehyde  overnight and embedded in paraffin. Paraffin sections were stained with Alexa Fluor 647–conjugated 1C10-1F7 mAb, DAPI (BioLegend), and H&E. Frozen thymus from 0-d-old WT mice was embedded in OCT compound (Sakura Finetek) and frozen sections were fixed with acetone. Frozen sections were stained with Alexa Fluor 647–conjugated 1C10-1F7 mAb and ER-TR5 mAb followed by Alexa Fluor 488–conjugated anti-rat IgG (Thermo Fisher Scientific). For multicolor confocal analysis, slides were mounted in ProLong Gold Antifade reagent (Invitrogen) and analyzed with a Zeiss LSM700 confocal microscope (Carl Zeiss). H&E–stained slide was analyzed with All-in-One Fluorescence Microscope BZ-9000 (Keyence).7 BM cells or 5 × 106 FL cells were intravenously injected into lethally irradiated (10 Gy) recipient 8-wk-old WT mice (Ly5.1/5.1). After 8 wk, reconstitution was confirmed.BM cells were extracted from 8-wk-old WT mice (Ly5.2/5.2) by flushing femurs and tibias and were then depleted of T cells using anti-CD3 mAb  and anti-rat IgG Dynabeads (Invitrogen). FL cells were extracted from the liver of embryonic day (ED) 14 WT mice (Ly5.2/5.2). 2 × 10M. bovis BCG (Tokyo strain) was purchased from Kyowa Pharmaceuticals and dissolved in 7H9 broth (Difco) supplemented with albumin–dextrose–catalase enrichment (BD Biosciences). Single colonies were grown with vigorous shaking at 37°C in Middlebrook 7H9 broth supplemented with 10% albumin–dextrose–catalase, 1% glycerol (Sigma-Aldrich), and 0.5% Tween 80 (Wako) until the optical density at 600 nm (OD600) reached 1. Bacteria were stored at −80°C in 50% glycerol as single-use aliquots. Mice were intratracheally infected with 1 × 106 CFUs of M. bovis BCG (Tokyo strain).Lyophilized K. pneumoniae ATCC strain 43,816, serotype 2 (ATCC) was grown in Difco Nutrient Broth (Difco) for 18 h at 37°C with vigorous shaking. Bacteria were pelleted by centrifugation and stored at −80°C in 50% glycerol as single-use aliquots. 3-wk-old WT mice were intraperitoneally injected with 200 μg of 1C10-1F7 or mouse IgG1 isotype control mAbs  on −3 d, −1 h, and 3 d. These mice were intranasally inoculated with K. pneumoniae at 1 × 103 CFUs on day 0.8-wk-old WT mice were intraperitoneally injected with 200 μg of 1C10-1F7 or mouse IgG1 isotype control mAb on days −3, 0, and 3. These mice were applied daily a topical dose of 62.5 mg of commercially available IMQ cream  on the shaved back for five consecutive days, translating to a daily dose of 3.125 mg of the active compound. Paraffin-fixed back skin of IMQ treatment on day 5 was fixed with phosphate-buffered 4% paraformaldehyde, embedded with paraffin, and stained with H&E. The back skin thickness was measured on days 0–5. The erythema score or scaling score of the back skin was scored on days 0–5 on a scale from 0 to 4.t test was used when only two groups were compared, and the survival curve was assessed by the log-rank test. P values <0.05 were considered to represent significant differences.Statistical significance was evaluated using Prism software (GraphPad). The


In the crystal, N—H⋯O hydrogen bonds involving the pyrrole amine and the ketone carbonyl O atoms link the molecules into [100] ribbons which form offset stacks along the  11H9NOS, was obtained in an improved yield compared to previous literature methods. The molecule is essentially planar with a maximum deviation of 0.085 Å from the mean plane through all non-H atoms. There is directive intermolecular hydrogen bonding in the form of N—H⋯O hydrogen bonds with a distance of 2.889 (3) Å between the pyrrole amine and the ketone carbonyl O atom. The resulting hydrogen-bonding network defines a ribbon parallel to the a axis. These ribbons form offset stacks along the b axis.The title compound, C The absorption and emission in an aza-BODIPY is shifted more towards the near infra-red region compared to BODIPY  and is almost planar in its crystalline form  of the pyrrole ring is protonated. Atom N1 is oriented opposite to the sulfur atom S1 of the thiophene ring to enable intermolecular hydrogen bonding  Å between the centroids of adjacent pyrrole or thiophene rings and a plane shift distance of 1.89 (3) Å between any two molecules in the three-dimensional crystal structure. C—H⋯O interactions also occur.Hydrogen bonding dominates the crystal packing of en Fig. 2, linkingrn Fig. 3. The anget al., 20161 (Table 2E)-1,3-di(thiophen-2-yl)prop-2-en-1-one  while the other is more planar  Å. This bonding network results in three separate sheets, parallel to the a-axis. A second non-classical hydrogen-bonding network [C—H⋯O = 2.381 (4) Å] is observed extending along the c-axis direction, generating a staggered ribbon. The combination of these two networks gives rise to a three-dimensional structure.A search of the Cambridge Structural Database -E)-1,3-Di(furan-2-yl)prop-2-en-1-one  in SANRIJ A and C18 (−0.152 Å) in SANRIJ B. Again, a non-classical hydrogen bonding network exists [C—H⋯O = 2.473 (18) Å] between aryl C—H atoms and the carbonyl oxygen. Each molecule participates in two hydrogen bonds and the network extends in a linear fashion along the b-axis direction, forming a network structure.(via an elimination unimolecular conjugate base (E1cB) reaction between 2-pyrrole-carbaldehyde  and 2-acetylthiophene  in 1:1 MeOH:H2O (10 ml) using NaOH  as a base. The resulting precipitate was filtered and then crystallized using a solution of CHCl3, layered with hexane to give a single crystal suitable for X-ray diffraction. [C11H9NOS]: yield 85% m.p 420–430 K.The title compound was synthesized 1H NMR : δ 6.34 , 6.74 , 7.01 , 7.06–7.10 , 7.15 , 7.63 , 7.81 , 7.83 , 9.17 . 13C NMR : δ 111.54, 115.16, 123.30, 128.15, 129.16, 131.11, 133.17, 133.98, 145.89, 182.05. HRMS (ESI): m/z calculated for C11H9NOS: 204.047690 (M + H)+. Found: 204.04776.Uiso(H) = 1.2 Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018012331/tx2008sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018012331/tx2008Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018012331/tx2008Isup3.cmlSupporting information file. DOI: 1864793CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


This makes the C1H1 unit a strong hydrogen-bond donor and results in the most striking feature of the structure °, in which the plane of the aryl ring almost eclipses the C1—C2 connectivity.H atoms bound to C in re Fig. 1, the intortho-F-aryl)-closo-1,2-C2B10H11] species to have been studied crystallographically is that with a 2′-fluoro-4′-(9′′-phenanthrenyl) substituent  Å, between the two crystallographically independent molecules in the asymmetric fraction of the unit cell, although the situation is somewhat complicated by partial disorder of both F atoms. The C1—C2 distance in , 1.660 (2) Å, stands good comparison with that in  , ,  and   species -closo-1,2-C2B10H11]  was dissolved in a 1:1 mixture of toluene and diethyl ether (40 mL). The colourless solution was cooled to 273 K before n-BuLi  was added dropwise over the course of 2 min. whilst stirring vigorously. The solution was warmed to room temperature and changed from colourless to yellow. After further stirring for 1 h the solution was cooled to 273 K, resulting in a white suspension. Whilst stirring vigorously, octafluorotoluene  was added dropwise over the course of 1 min., causing the solution to turn from yellow to deep red. The solution was stirred for 4 h at room temperature and then quenched with saturated [NH4]Cl . The organic layer was isolated and the aqueous phase extracted with Et2O (3 × 20 mL). The organic phases were combined and reduced in volume in vacuo to yield a brown residue. Products were isolated by column chromatography on silica eluting with 313–333 K petroleum ether to give both the target compound   and the disubstituted species  : 4.88 . 11B{1H} NMR : −0.32 (1B), −1.80 (1B), −8.06 (2B), −9.62 (2B), −11.17 (2B), −12.89 (2B). 19F NMR : −56.72 , −135.17 , −137.26 . Crystals of  suitable for a single-crystal X-ray diffraction study were grown from the slow evaporation of a 313–333 K petroleum ether solution of the product.CVertex–Centroid Distance  = 1.2Ueq(B or C). Five poorly fitting reflections were omitted which marginally decreased the R-factor and standard uncertainties from the previous refinement.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019004067/lh5894sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019004067/lh5894Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019004067/lh5894Isup3.molSupporting information file. DOI: 1905663CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal structure of the title compound, C—H⋯O and C—H⋯N hydrogen bonds as well as C—H⋯π(ring) interactions link individual molecules into a three-dimensional network. 19H16N4O, the planar pyrazolopyrimidine moiety is inclined to the attached phenyl rings by 35.42 (4) and 54.51 (6)°. In the crystal, adjacent molecules are linked into chains parallel to [110] and [1In the title molecule, C The attached benzene rings (C6–C11 and C13–C18) are inclined to the above plane by 35.42 (4) and 54.51 (6)°, respectively.B⋯O1 hydrogen bonds between a methyl H atom and the methoxy O atoms of adjacent molecules lead to the formation of chains extending alternately parallel to [110] and pyrimidine , 4-iodoanisole , Cs2CO3 , K3PO4 , 1,10-phenanthroline , and Pd(OAc)2  in DMA (3 ml) was heated to 438 K for 48 h. After completion of the reaction, the mixture was allowed to cool to room temperature and the solvent was removed under reduced pressure. Water (15 ml) was added, and the resulting aqueous phase was extracted with CH2Cl2 (3 × 15 ml). The combined organic layers were dried with MgSO4 and concentrated under vacuum. The residue was purified by column chromatography on silica gel (EtOAc/petroleum ether). The title compound was recrystallized from ethanol at room temperature, giving colorless crystals .Under an atmosphere of argon, a mixture of 1-methyl-4-phenyl-1Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019004894/wm5499sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989019004894/wm5499Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019004894/wm5499Isup3.cmlSupporting information file. DOI: 1909062CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A new macrocycle obtained by the Michael reaction of azacrown ether with dimethyl acetylenedicarboxylate was studied by X-ray diffraction, and its potential bioactivity was estimated from the structural data. 30H34N2O9 (4), is a product of the Michael reaction of azacrown ether with dimethyl acetylenedicarboxylate modified by an addition of NH3 (aq.) at 298 K. The aza-14-crown-4-ether ring adopts a bowl conformation. The dihedral angle between the planes of the benzene rings fused to the aza-14-crown-4-ether moiety is 8.65 (5)°. The tetrahydropyridine ring has a boat conformation. The molecular conformation is supported by one N—H⋯O and two C—H⋯O intramolecular hydrogen bonds. Both heterocyclic and amino N atoms have essentially planar configurations (sums of the bond angles are 359.35 and 358.00°). Compound 4 crystallizes as a racemate consisting of enantiomeric pairs of the 1R,21S diastereomer. In the crystal, molecules of 4 are connected by N—H⋯O hydrogen bonds, forming chains along [100]. According to the PASS program , compound 4 may exhibit antiallergic (72% probability) and antiasthmatic (67%) activity, as well as be a membrane permeability inhibitor (65%).The title compound, C The dihedral angle between the planes of the benzene rings fused to the aza-14-crown-4-ether moiety is 8.65 (5)°. The tetrahydropyridine ring adopts a boat conformation. The conformations of the aza-14-crown-4-ether and piperidine rings are supported by the three intramolecular (one N—H⋯O and two C—H⋯O) hydrogen bonds , 3.14 g (10.00 mmol) of 1,5-bis-(2-formylphenoxy)-3-oxaopetane (2) and 1.00 g (13.00 mmol) of ammonium acetate in a mixture of 30 ml ethanol and 1 ml acetic acid was stirred at 298 K. The reaction was monitored by TLC and found to be complete after 6 h. The reaction mixture was allowed to cool to room temperature before being neutralized with sodium carbonate solution; the product was then extracted with chloroform (3 × 50 ml). By TCL, compound 3 was determined to be successfully synthesized. The solvent (CDCl3) was evaporated under vacuum until 30ml of CDCl3 was left, 1.42 g (10 mmol) of DMAD was added and the solution was stirred for 30 minutes at 298 K. Then NH3 (aq.) was added to the reaction mixture, which was stirred continuously. After three days, the residue was purified by column chromatography and recrystallized from ethanol to obtain 2.27 g of the pure azacrown ether 4 as light-yellow crystals (yield 60%). Tm = 525–526 K. Rf = 0.85 [n-hexane:ethyl acetate = 1:1 (v:v)].A solution of 1.30 g (10.00 mmol) of ethyl acetoacetate (Uiso(H = 1.2Ueq(N)]. Other hydrogen atoms were placed in calculated positions with C—H bond lengths of 0.95–1.00 Å and refined using a riding model with constrained isotropic displacement parameters [Uiso(H) = 1.5Ueq(C) for the CH3 groups and 1.2Ueq(C) for all others].Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901801160X/ld2144sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698901801160X/ld2144Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901801160X/ld2144Isup3.cmlSupporting information file. DOI: 1862265CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the title compound, the seven-membered diazepine ring adopts a boat-shaped conformation. In the crystal, molecules are linked by pairs of N—H⋯O hydrogen bonds, forming inversion dimers with an  10H8Cl2N2O, the seven-membered diazepine ring adopts a boat-shaped conformation. The mean planes of the two rings of the benzodiazepine unit are inclined to each other by 22.05 (6)°. In the crystal, molecules are linked by pairs of N—H⋯O hydrogen bonds, forming inversion dimers with an R22(8) ring motif. The dimers are linked by C—H⋯π interactions, forming layers lying parallel to (10In the title compound, C The mean planes of the two rings are inclined to each other by 22.05 (6)°. The C9=N2 bond has a Z configuration and a bond length of 1.2737 (18) Å. The C1—N2 [1.4124 (17) Å] and C6—N1 [1.4068 (18) Å] bond lengths are typical for a 2,3-dihydro-1H-1,5-benzodiazepin-2-one ring system and similar to those observed for the structure of a very similar compound, 4-methyl-2,3-dihydro-1H-15-benzodiazepin-2-one monohydrate . In all 12 compounds, the diazopine ring has a boat-shaped conformation, as does the title compound. The benzene ring and the mean plane of the diazepine ring are inclined to each other by dihedral angles ranging from ca 19.95 to 29.16°, compared to 22.05 (6)° in the title compound. The C=O bond lengths vary from ca 1.217–1.241 Å and the C=N bond lengths vary from ca 1.272–1.295 Å. In the title compound, the corresponding bond lengths are 1.2288 (18) and 1.2737 (18) Å, respectively. The Caromatic—N bond lengths in the diazepine ring range from ca 1.391 to 1.415 Å, compared to values of 1.4124 (17) and 1.4068 (18) Å for bonds C1—N2 and C6—N1, respectively, in the title compound. Hence, the various geometrical parameters mentioned above for the title compound are typical for 2,3-dihydro-1H-1,5-benzodiazepin-2-ones. In the crystals of all but one compound, molecules are linked by pairs of N—H⋯O hydrogen bonds, forming inversion dimers with an A search of the Cambridge Structural Database  to 1.092 (blue) Å using the CrystalExplorer program  surface resolution with the three-dimensional de + di = 2.5 Å and two symmetrical points at the top, bottom left and right. These data are characteristic of C—H⋯O hydrogen bonding.Fig. 6di, de) points associated with hydrogen atoms. It is characterized by an end point that points to the origin and corresponds to di = de = 1.08 Å, which indicates the presence of the H⋯H contacts in this study, which make a contribution of 54.3% to the crystal packing. The C⋯H/H⋯C graph in Fig. 7vice versa and has two symmetrical wings on the left and right sides (6.8%). Much weaker C⋯C (5.5%), O⋯N/N⋯O (2.4%), O⋯O (0.3%) and S⋯H/H⋯S (0.2%) contacts also occur.The H⋯H graph in Fig. 7A view of the three-dimensional Hirshfeld surface of the title compound plotted over electrostatic potential energy in the range −0.082 to 0.042 a.u. using the STO-3G basis set at the Hartree–Fock level of theory is shown in Fig. 8Z)-4-(2-oxopropylidene)-4,5-dihydro-1H-benzo [b]-diazepine-2(3H)-one under phase-transfer catalysis (PTC) conditions using tetra-n-bromide butylammonium (TBAB) as catalyst and potassium carbonate as base.The title compound was synthesized by the reaction of dichloromethane with -one (2.87 mmol) in dichloromethane (30 ml) as reagent and solvent, potassium carbonate (5.71 mmol) and a catalytic amount of tetra-n-butylammonium bromide (0.37 mmol) were added. The mixture was stirred for 48 h. The solid material was removed by filtration and the solvent evaporated under vacuum. The residue was purified through silica gel column chromatography using hexane/ethyl acetate (ratio 8:2). Slow evaporation at room temperature lead to the formation of colourless single crystals (yield 69%).To a solution of 4-(2-oxopropylidene)-4,5-dihydro-Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901801681X/xu5953sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S205698901801681X/xu5953Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901801681X/xu5953Isup4.cmlSupporting information file. DOI: 10.1107/S205698901801681X/xu5953sup3.pdfCSD search results. DOI: 1881324CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II atom, located on a twofold rotation axis, is coordinated by three chelating ligands, leading to a distorted octahedral CdN4O2 coordination sphere.In the crystal, the Cd 16H16O3)2(C12H8N2)]·4H2O, the Cd ion lies on a twofold rotation axis and is chelated by two monodeprotonated residues of the l-tyrosine-derived ligand (S)-2-[(2-hydroxybenzyl)amino]-3-(4-hydroxyphenyl)propanoic acid (L) in a κ2N,O amino acid chelating mode, exhibiting an -trans disposition, and by 1,10-phenanthroline in a κ2N,N′ mode. The latter ligand is also located about the twofold rotation axis that bisects the central six-members ring. The phenolic groups remain protonated and non-coordinating and take part as acceptors in the intramolecular hydrogen bonds with the amino groups of the acido ligands. The Cd ion is six-coordinated in a distorted octahedral environment. In the crystal, O—H⋯O hydrogen bonds result in the formation of three-dimensional network structures. The title complex has also been characterized by IR and 1H NMR spectroscopy and DFT studies. The crystal studied was refined as an inversion twin.In the title compound, [Cd(C In recent years they have found applications in the fields of analytical chemistry, medicine and biological processes, displaying antifungal, antibacterial and anticancer activities (Przybylski L-H)2(phen)] and two water molecules of solvation -trans disposition. The Cd—O and Cd—N bond lengths are similar, being 2.325 (5), 2.335 (6) and 2.323 (6) Å for Cd1—O4, Cd1—N1 and Cd1—N2, respectively. All three ligands form five-membered chelate rings. Unlike the chelate ring formed by the phenanthroline ligand which is virtually planar, the one created by the L residue exhibits an envelope conformation with the deviation of the Cd atom from the mean plane defined by the other four atoms being 1.0692 (3) Å. The N—Cd—O and N—Cd—N bite angles are 70.5 (2) and 72.0 (4)°, respectively. The phenolic O—H group remains protonated and non-coordinating, albeit participating in an extensive intermolecular hydrogen-bonding network. An intramolecular N1—H1⋯O1 hydrogen bond  calculations were performed to investigate the electronic structure and characteristic vibrations. The calculated frequencies were found within the range, shown in Table 2E) between the HOMO and LUMO values are more polarizable, thereby acting as soft molecules with a higher chemical reactivity. However, complexes with large energy gap offer greater stability and lower chemical reactivity than those with a small HOMO–LUMO energy gap. The DFT study revealed that the HOMO, HOMO-1, HOMO-2 and HOMO-3 energies are localized on the N1, N4, O2, O3, O6, O7, C8, C9, C35, C36 and C37 atoms of the amino acid ligand, partially localized on the Cd centre, namely dx2 − y2, as shown in Fig. 4The LUMO and HOMO orbital energy parameters are accountable to a significant extent for the charge transfer, chemical reactivity and kinetic/thermodynamic stability of a molecule. Metal complexes with a small energy gap  and curvedness (−4.0 to 0.4 a.u.). The dnorm surface has a red–white–blue colour scheme, whereas deep-red spots highlight shorter contacts i.e. hydrogen bonding. The white areas represent contacts around the van der Waals separation, such as H⋯H contacts, and the blue regions are devoid of such close contacts. On the Hirshfeld surface mapped with the shape-index function, one can examine both red regions corresponding to C—H⋯π interactions as well as ‘bow-tie patterns’, which indicate the presence of aromatic stacking (π–π) interactions. The curvedness surface indicates that the electron density of the surface curves around the molecular interactions. The fingerprint plots, presented in Fig. 6The Hirshfeld surfaces of the title compound are illustrated in Fig. 5et al., 2016II ion coordinated by 2-hydroxybenzyl derivatives of amino acids yielded only one hit -d,l-aspartic acid. In this complex, the doubly deprotonated (by the phenolic and β-carboxylic groups) residue of the ligand is coordinated in an -tridentate mode including the phenolic oxygen, unlike the title compound in which the phenolic group is non-coordinating. The second oxygen atom of the β-carboxylic group bridges the neighboring mononuclear Cd units into a one-dimensional chain. In addition, there are few structures of complexes with zinc or cadmium analogues  with 2-hydroxybenzyl derivatives of alanine. In all these complexes, the ligand is also coordinated in an -tridentate manner, with an additional μ2-function of the phenolic oxygen, which results in the formation of a Zn2O2 binuclear core in all cases -2-[(2-hydroxybenzyl)amino]-3-(4-hydroxyphenyl)propanoic acid (L)o-salicylaldehyde  was added dropwise to a stirring solution of l-tyrosine  and LiOH·H2O  in methanol (25 mL). Stirring was continued for 2 h, followed by the addition of sodium borohydride  with further stirring for 1 h. The solvent was evaporated and the resulting sticky mass was dissolved in water and acidified with dilute HCl. The pH of the solution was maintained between 5–7. The ligand precipitated as a brown solid. It was washed thoroughly with water and MeOH after filtration and dried in a vacuum desiccator. Yield 1.60 g (76%). m/z  379.087 . IR , ν(COO)asym 1579 (s), ν(COO)sym 1394 (s). 1H NMR : Hcp.o , Hcp.o , Hcp , Hcp.m , Ha , Ha,c′ , Hb , Hc , Hc,c′ , Hd,d′ , He,e′ . As a result of geminal coupling, Hc split into two non-equivalent Hc and Hc′.A methanolic solution of 2(phen)]·4H2OSynthesis of ·4H2O (2). Yield 49%. [Cd(L)2(phen)]·4H2O: IR  ν(COO)assym 1651, ν(COO)sym 1381, ν 1250. 1H NMR [Cd(L)2(phen)]·4H2O] : 2.4 , 2.5 , 2.8 , 3.1 , 3.4 , 6.1 , 6.5 , 7.0 , 7.9 , 8.0 , 8.7 , 9.0 . ESI–Mass (-ve) 929.2 .A methanolic solution of Cd(NOUiso(H) = 1.5Ueq(O). All C-bound H atoms were positioned geometrically and refined using a riding model with C—H = 0.93 Å and with Uiso(H) = 1.2Ueq(C). The crystal studied was refined as an inversion twin.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S205698901801157X/xu5935sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S205698901801157X/xu5935Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901801157X/xu5935sup3.gif1487 +COO symmetrical stretch. DOI: Click here for additional data file.10.1107/S205698901801157X/xu5935sup4.gif1237 +C-N stretch. DOI: Click here for additional data file.10.1107/S205698901801157X/xu5935sup5.gif885 +NH wagging. DOI: Click here for additional data file.10.1107/S205698901801157X/xu5935sup6.gif756 +CH rocking. DOI: Click here for additional data file.10.1107/S205698901801157X/xu5935sup7.gif1598 +COO antisymmetrical stretch. DOI: Click here for additional data file.10.1107/S205698901801157X/xu5935sup8.gif3414 +NH stretching. DOI: 1534110CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


However, β2M-mediated signaling in ER+ and ER− breast cancer with HER2− remains unclear.Previous studies have demonstrated that β2-microglobulin (β2M) promotes the growth and survival of a variety of cancer cells and has different regulatory effects on the expression of Bcl-2 and HER2 in HER2− breast cancer cells, and the possible relevant signaling molecules were subsequently analyzed by real-time PCR and western blotting. These signaling molecules were also analyzed by real-time PCR and immunohistochemistry (IHC) in two types of HER2− breast cancer tissues, and the associations between β2M and these signaling molecules were assessed using Spearman’s correlation analysis.β2M expression vector and siRNA were transfected into two types of HER2+ breast cancer cells with HER2−. β2M silencing upregulated p-CREB/CREB and VEGF protein and significantly downregulated p-ERK/ERK levels, and β2M overexpression downregulated p-CREB/CREB and VEGF, significantly upregulated p-ERK/ERK levels, and both resulting processes did not affect HIF-1α and SGK1 signaling in ER− breast cancer cells with HER2−. β2M expression was positively correlated with p-CREB, p-SGK1, and Bcl-2 expression and had no correlation with HIF-1α, VEGF, and p-ERK1/2, whereas p-SGK1 exhibited a significantly positive correlation with Bcl-2 expression in cancer tissues of patients with luminal A breast cancer, which coincide with the results obtained from the same molecular types of breast cancer cells except CREB signaling. However, β2M expression did not show a significant correlation with HIF-1α, p-CREB, VEGF, p-SGK1, p-ERK1/2, and Bcl-2 expression in cancer tissues of patients with basal-like breast cancer, which was discordant with the results obtained from the same molecular types of breast cancer cells.β2M silencing downregulated p-SGK1/SGK1 levels and Bcl-2 expression, and β2M overexpression downregulated p-CREB/CREB and significantly upregulated p-SGK1/SGK1 levels and Bcl-2 expression, and both resulting processes did not affect HER2, HIF-1α, VEGF, and ERK signaling in ER+ and ER− breast cancer with HER2−, and it may promote tumor survival through the SGK1/Bcl-2 signaling pathway in ER+ breast cancer with HER2− and has no regulatory effects on ER− breast cancer with HER2−.β2M has a different molecular regulatory mechanism between ER Its serum expression levels are elevated in cancer patients, including patients with breast cancer, thyroid cancer, liver cancer, small cell lung cancer, renal cell carcinoma, prostate cancer, and gastrointestinal cancers . Previou+), PR-positive (PR+), and HER2-negative (HER2−) status, whereas the β2M siRNA significantly upregulated the Bcl-2 and HER2 expression in breast cancer cells MDA-MB-231 with ER-negative (ER−), PR-negative (PR−), and HER2− status [+ and ER− breast cancer cells with HER2− [+ and ER− breast cancer with HER2−.Some studies have shown that metastatic breast cancer is likely associated with a β2M/creatinine ratio > 3.8 , and pat− status . Thus, t− status . Therefo+ PR+ HER2−), T47D (ER+ PR+ HER2−), MDA-MB-231 (ER− PR− HER2−), and Hs578T (ER− PR− HER2−) were obtained from the Cell Bank of the Chinese Academy of Sciences  and cultured in high glucose Dulbecco’s modified Eagle’s medium  with 10% heat-inactivated fetal bovine serum  at 37 °C with 5% CO2.The human breast cancer cell lines MCF-7  were obtained from the Department of Pathology, and serial 4-μm sections were processed for the IHC. The tumor and adjacent fresh tissues  were obtained from surgical specimens resected from patients without chemotherapy and radiotherapy before operation, immediately frozen in liquid nitrogen and stored at − 80 °C for preparation of total RNA.The sequence-specific β2M siRNA (siβ2M) and scrambled siRNA were purchased from GenePharma Co., Ltd. . The sequences of the siRNAs are shown in Table KpnIand BamHIcloning sites. The β2M primer sequences were as follows: forward, 5′-CGGGGTACCATGTCTCGCTCCGTGGCCTT-3′; reverse, 5′-CGCGGATCCTTACATGTCTCGATCCCACT-3′. The β2M cDNA was inserted between the KpnIand BamHIsites in the pEGFP-C1 vector. The recombinant vector was designated pEGFP-C1-β2M, which was subsequently extracted with an E.Z.N.A. Endo-Free Plasmid Mini Kit . Plasmid concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific) at a wavelength of 260 nm, and purity was checked by measuring the 260/280 and 260/230 ratios. Transfection was conducted using Lipofectamine 3000 (Thermo Fisher Scientific) as recommended by the manufacturer. Briefly, cells were seeded in six-well plates at a density of 1.5–3 × 105 per well. After 24 h, cells were transfected using 2.5 μg of pEGFP-C1-β2M and 3.75 μL Lipofectamine 3000 per well, medium was changed after 6 h. The empty pEGFP-C1 vector was used as control. At approximately 36 h post-transfection, the cells were lysed and analyzed using real-time PCR and western blotting.To construct the β2M expression vector, β2M cDNA was isolated by reverse transcription-PCR (RT-PCR) from cells and flanked with -ΔΔCt method, with the Ct values normalized using GAPDH as the internal control.Total RNA was extracted from the cultured cells, tumor tissues, and adjacent tissues using RNAiso Plus . For each sample, 500 ng of total RNA was reverse transcribed using PrimeScript RT Master Mix (Takara), following the manufacturer’s instructions. The cDNA was subjected to real-time PCR analysis in a Rotor-gene 3000 (Corbett Research) using SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara). The cycling conditions were as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. The primer sequences were designed and synthesized by Takara. All of the primer sequences are presented in Table Cells were trypsinised and centrifuged. The pellets were then resuspended in cell lysis buffer that contained phenylmethylsulfonyl fluoride, a protease inhibitor, and a phosphatase inhibitor . Approximately 40 μg proteins per lane were separated in 12% SDS-PAGE gels and transferred onto nitrocellulose membranes. After blocking with TBST containing 5% nonfat milk powder, the membranes were incubated with primary antibodies against β2M , HIF-1α , CREB , phospho-CREB (Ser133) , VEGF , HER2 , ERK1/2 , phospho-ERK1/2 (Thr202/Tyr204) , serum and glucocorticoid-regulated kinase 1 , phospho-SGK1 (Thr256) , and Bcl-2  at 4 °C overnight. After washing with TBST, the membranes were incubated with the corresponding secondary antibodies, which were conjugated with horseradish peroxidase (Santa Cruz Biotechnology). Immunoreactive bands were visualized with SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific) and recorded with a ChemiDoc™ XRS+ (Bio-Rad). β-actin was used as internal control. Signal intensities were subsequently quantified using Image Lab quantification software .Formalin-fixed, paraffin-embedded tissue specimens were obtained and handled by standard surgical oncology procedures. Serial 4-μm sections were prepared and stained using biotin-streptavidin HRP detection systems  with the following primary antibodies: β2M , HIF-1α , VEGF , p-CREB , p-ERK1/2 , p-SGK1 , and Bcl-2 . Immune complexes were visualized using a 3,3′-diaminobenzidine tetrahydrochloride substrate solution (ZSGB-BIO). The slides were then counterstained with hematoxylin and mounted. Controls were prepared by omitting the primary antibodies.IHC staining was evaluated independently by two authors with reference to both the staining intensity and the extent of positively stained area. Staining intensity was scored as follows: 0 (negative), 1 (weak), 2(moderate), or 3 (strong). Extent of staining was scored as 0 (0%), 1 1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%) according to the percentages of the positively staining areas in relation to the whole carcinoma area. The sum of the intensity and extent scores was used as the immunohistochemical score (IHS), which ranged from 0 to 7. For the purpose of statistical analysis, tumors having a final staining score of ≥3 were considered positive [%, 2 (26–p < 0.05 (*) or p < 0.01 (**) were considered statistically significant.Statistical comparisons were performed using SPSS version 23.0. Data of the silencing and overexpression experiments were presented as the mean ± SD and analyzed by the independent-samples t test. Wilcoxon matched-pair signed-rank test was used to analyze differences in mRNA and protein expression of signaling molecules between breast cancer tissues and their matched adjacent tissues. Spearman correlation analysis was conducted to examine the strength of relationship between protein expression. + and ER− breast cancer cell lines with HER2−, the β2M gene was silenced, and possible relevant signaling molecules were analyzed by real-time PCR and western blotting. The ER+ and ER− cells were transiently transfected with siβ2M which had a significant effect on downstream genes in our previous study [p < 0.01; left panels of Fig. p < 0.01; middle and right panels of Fig. p < 0.01), but had no effects on the mRNA levels of HER2 and VEGF (p > 0.05) in ER+ MCF-7 cells. At the protein levels, p-CREB/CREB, p-SGK1/SGK1, and Bcl-2 were significantly reduced (p < 0.01), whereas those of HER2, HIF-1α, VEGF, and p-ERK/ERK did not change (p > 0.05). In the T47D cells , whereas p-CREB/CREB significantly increased following β2M silencing (p < 0.01). These results suggest that β2M silencing downregulated p-SGK1/SGK1 levels and Bcl-2 expression, but did not affect the HER2, HIF-1α, VEGF and ERK signaling in ER+ breast cancer cells with HER2−. Additionally, changes in p-CREB/CREB levels were discordant following β2M silencing.To determine the regulatory effects of β2M silencing between ERus study  or contr− MDA-MB-231 cells , VEGF (p = 0.000) and Bcl-2 (p = 0.040) and significantly reduced HIF-1α mRNA level (p = 0.001). In terms of protein expression levels, HER2, p-CREB/CREB, VEGF, and Bcl-2 were significantly enhanced (p < 0.01), whereas HIF-1α and p-SGK1/SGK1 did not change (p > 0.05), and p-ERK/ERK was significantly reduced following β2M knockdown (p < 0.01). In the ER− Hs578T cells, the changes in the expression of HIF-1α, p-CREB/CREB, p-SGK1/SGK1, and p-ERK/ERK were similar to those in siβ2M-transfected MDA-MB-231 cells, whereas siβ2M imparted opposite effects on the expression of HER2 and Bcl-2  or pEGFP-C1 (control) for 36 h, and then analyzed the possible relevant signaling molecules described above by real-time PCR and western blotting. Figure p < 0.01; left panels of Fig. p < 0.01; middle and right panels of Fig. + MCF-7 cells  and significantly increased the mRNA levels of HIF-1α and Bcl-2 (p < 0.01). Western blotting analysis revealed that there were no significant differences in the expression levels of HER2, HIF-1α, VEGF, and p-ERK/ERK (p > 0.05), p-SGK1/SGK1 and Bcl-2 were markedly upregulated (p < 0.01), and p-CREB/CREB was downregulated (p < 0.05) following β2M overexpression. In ER+ T47D cells , VEGF (p = 0.003), and Bcl2 (p = 0.000) and significantly increased HIF-1α mRNA levels (p = 0.001). Western blotting analysis also showed that β2M overexpression downregulated HER2 (p = 0.043), p-CREB/CREB (p = 0.023), VEGF (p = 0.02), and Bcl-2 (p = 0.006), significantly upregulated p-ERK/ERK (p < 0.01), and did not affect HIF-1α and p-SGK1/SGK1 (p > 0.05). Consistent with the findings in MDA-MB-231 cells, HIF-1α, p-CREB/CREB, VEGF, p-SGK1/SGK1, and p-ERK/ERK presented similar variations in β2M-overexpressed Hs578T cells. However, β2M overexpression imparted the opposite effects on the expression of HER2 and Bcl-2 in the Hs578T cells. These results suggest that β2M overexpression downregulates p-CREB/CREB and VEGF levels and significantly upregulates p-ERK/ERK levels, but does not affect HIF-1α protein and p-SGK1/SGK1 levels in the cell lines of ER− breast cancer with HER2−. Additionally, changes in HER2 and Bcl-2 levels were discordant following β2M overexpression.In ER− breast cancer tissues were examined by real-time PCR and IHC. Real-time PCR analysis did not reveal any significant differences in the mRNA levels of β2M, HER2, HIF-1α, VEGF, and Bcl-2 between the cancer tissues and adjacent tissues , and no significant differences in expression of HIF-1α, p-CREB, p-SGK1, and p-ERK1/2 were detected between cancer tissues and their matched adjacent tissues , p-SGK1 , and Bcl-2  and had no correlation with HIF-1α, VEGF, and p-ERK1/2 (p > 0.05) in cancer tissues of patients with luminal A breast cancer. Furthermore, a significant positive correlation was observed between p-SGK1 and Bcl-2  (Table p > 0.05), except that VEGF showed a strong positive correlation with Bcl-2  in cancer tissues of patients with basal-like breast cancer  P3, which could induce phosphorylation of SGK1 at its hydrophobic motif (Ser422) that in turn promotes the interaction of SGK1 with phosphoinositide-dependent kinase 1 (PDK1). Then, PDK1 activates SGK1 by phosphorylating the T-loop residue (Thr256) [+ MCF-7 and T47D cells [+ breast cancer cells with HER2− (MCF-7 and T47D). Second, β2M inhibits CREB signaling and the expression of VEGF protein and activates ERK signaling, but does not affect HIF-1α and SGK1 signaling in ER− breast cancer cells with HER2−. CREB, a transcription factor, is involved in the tumorigenicity of HER2/Neu-overexpressing tumor cells [− breast cancer cells with HER2−. The reason behind the observed opposite regulatory effects of β2M on ER− breast cancer cells with HER2− may be that the triple-negative breast cancer is a special type of breast cancer that has special biological behavior and a very complex regulatory mechanism. Third, β2M exhibits different regulatory effects on CREB and p-CREB in the cell lines of ER+ breast cancer with HER2− (MCF-7 and T47D), and HER2 and Bcl-2 in the cell lines of ER− breast cancer with HER2− (MDA-MB-231 and Hs578T), which may be due to the fact that breast cancer has a very complex molecular regulatory mechanism, and every type of breast cancer has several subtypes, and these signaling molecules may be regulated by other pathways in corresponding subtypes of breast cancer. Fourth, β2M is positively correlated with p-CREB, p-SGK1, and Bcl-2 and has no correlation with HIF-1α, VEGF and p-ERK1/2, and p-SGK1 has a significantly positive correlation with Bcl-2 in cancer tissues of patients with luminal A breast cancer, which agrees with the results obtained from the same molecular types of breast cancer cells except CREB signaling. However, β2M expression shows no significant correlation with HIF-1α, p-CREB, VEGF, p-SGK1, p-ERK1/2, and Bcl-2 except for VEGF, which shows a strong positive correlation with Bcl-2 in cancer tissues of patients with basal-like breast cancer, thereby showing discordance with the results obtained from the same molecular types of breast cancer cells. One possible reason is that large differences may exist among cancer cell lines and tissue samples, particularly in terms of its molecular genome [+ breast cancer with HER2− and have no regulatory effects in ER− breast cancer with HER2−. It is possible that the regulatory function of β2M is correlated with ER expression in HER2− breast cancer. The regulatory mechanism of β2M in HER2− breast cancer needs to be further investigated.In this study, our results demonstrated the following. First, β2M activates SGK1 signaling and upregulates Bcl-2 expression, and do not affect HER2, HIF-1α, VEGF, and ERK signaling in ER(Thr256) . Subsequ7D cells . Therefoor cells , 33; VEGor cells –36. Actior cells  or overeor cells  in humanr genome , 38, andr genome . In addir genome . Therefo+ and ER− breast cancer with HER2−. β2M may promote tumor survival through the SGK1/Bcl-2 signaling pathway in ER+ breast cancer with HER2− and have no regulatory effects on ER− breast cancer with HER2−. Understanding the regulation of β2M-mediated signaling pathways will help to identify novel therapeutic targets for patients with different types of breast cancer.β2M has different molecular regulatory mechanisms between ER


In order to find new potential anticancer agent, Mucor polymorphosporus was used to transform cis-auraptene. Four new terpene coumarins with notable changes in the skeletal backbone, 2′-Z auraptene A-D (2–5), were obtained and evaluated for their antiproliferative effects against human normal gastric epithelium cells and human gastric cancer cells. These new compounds showed selective cytotoxic activity against MGC-803 cells with IC50 values from 0.78 ± 0.13 to 10.78 ± 1.83 μM and the therapeutic index could also be significantly improved (TI = 59.0) compared with that of 1 (TI = 5.5). The structures of these metabolites were elucidated through extensive spectroscopic methods, and the possible biotransformation pathway of 1 by Mucor polymorphosporus was also proposed. Furthermore, the mechanism of the antiproliferative effects against MGC-803 cells of the most potent compound, 2′-Z auraptene A (2), was characterized. Annexin V/PI staining and abnormal expression of apoptosis-related protein suggested that compound 2 induces apoptosis in gastric cancer MGC-803 cells. Therefore, it is possible that compound 2 has the potential to be applied in gastric cancer therapy.2′-Z auraptene (The online version of this article (10.1007/s12272-018-1028-0) contains supplementary material, which is available to authorized users. Gastric cancer is one of the most common malignancies in the world and characterized by its high mortality rate .Coumarins are a wide family of natural and synthetic compounds, which exhibited a wide range of pharmacological activities including antitumor , B (3), C (4), D (5) . High resolution electrospray ionization mass spectrometry (HR–ESI–MS) was performed on a Thermo Scientific LTQ-Obitrap XL . Phosphate Buffered Saline (PBS) and Dulbecco′s modified Eagle′s medium (DMEM) were provided by HyClone . Fetal bovine serum (FBS), penicillin, streptomycin, and Trypsin–EDTA were purchased from Solarbio . DMSO (cell culture grade) and 3--2, 5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich . The Annexin V-FITC Apoptosis Kit was supplied by KeyGEN Biotech . The cECL Western Blot Kit was provided by BD Biosciences . Antibodies against β-actin, Bax, Bcl-2 and cleaved caspase-3 were obtained from Santa Cruz Biotechnology . 7-hydroxycoumarin (≥ 98% purity), nerol (≥ 98% purity), PBr3 (≥ 95% purity), pyridine (≥ 98% purity), K2CO3 (≥ 99% purity), KI (≥ 99.9% purity) were supplied by Energy Chemical . All solvents used were obtained from Beijing Chemical Works .Pre-coated silica gel plates  were used to perform thin layer chromatography analysis. Sephadex LH-20  and silica gel  were used for column chromatography. Semi-preparative HPLC was performed on a K1001 analytic LC instrument  equipped with a K-2600 UV detector and a YMC-Pack ODS-A column . Nuclear magnetic resonance (NMR) spectra were recorded using Bruker AVANCE Ш 600 spectrometer  operating at 150 MHz for 1) was synthesized as the following methods , and appropriate pyridine in diethyl ether was placed in ice-water bath with a nitrogen atmosphere. After 0.5 h stirring, ice water was added to terminate reaction, and subsequently the mixture was extracted by diethyl ether to afford the rude intermediation used in the next step without further purification. Then, a mixture of the intermediation obtained from above, 7-hydroxycoumarin , K2CO3 , and KI  in acetone were stirred at room temperature for 2–3 h. Subsequently, the obtained crude mixture was purified by silica gel chromatography, eluting with petroleum ether- ethyl acetate .2′-Z auraptene  and identified by spectroscopic methods. NMR data of 1 was conform to that of published data for (Z)-7- oxy)coumarin (Coates and Melvin 1 as (Z)-7-oxy)-2H- chromen-2-one, and named it as cis-auraptene in our study.Compound m/z 297.1094 ; c 0.086, MeOH); 1H NMR data, see Table 13C NMR data, see Table Colorless solid; (+) HR-ESI–MS m/z 337.1406 ; c 0.163, MeOH); 1H NMR data, see Table 13C NMR data, see Table Colorless solid; (+) HR-ESI–MS m/z 353.1355 ; c 0.243, MeOH); 1H NMR data, see Table 13C NMR data, see Table Colorless solid; (+) HR-ESI–MS m/z 353.1356 ; c 0.083, MeOH); 1H NMR data, see Table 13C NMR data, see Table Colorless solid; (+) HR-ESI–MS Mucor polymorphosporus (CGMC3.3443), was obtained from the China General Microbiological Culture Collection Center. Mucor polymorphosporus was cultured on potato dextrose agar slants at 37 °C and stored at 4 °C. The whole biotransformation process were performed in potato medium, which was prepared according to the following methods: A mixture of distilled water (1000 mL) and mincing husked potato (250 g) was boiled for half an hour, and then was filtered. Subsequently, the filtrate was mixture with distilled water and glucose (20.0 g), and was autoclaved at 121 °C half an hour.The fugal strain, 1 (500.0 mg) dissolved in methanol was added to each flask with another 4-day incubation. Finally, the broth was extracted by an equal volume of ethyl acetate for three times, and the organic solvent was vacuum dried to obtain a rude mixture . Then, compound 2O on a Thermo C18 column , respectively. Finally, compound 2  was yielded from Fr. 2 eluting with MeOH–H2O (64:36) system; Compound 3  was obtained from Fr. 3 eluted with MeOH–H2O (65:35) system; Compound 4  was separated from Fr. 5 using a MeOH–H2O (57:48) system; Compound 5  was separated from Fr. 6 eluting with a MeOH–H2O (55:45) system.The crude extract (1.1 g), eluted with petroleum ether-ethyl acetate , was fractionated into nine fractions (Fr. 1–9) by chromatography using a silica gel column . These fractions were separated by semi-preparative HPLC eluted with MeOH–H2. Cells that used in experiments were passaged at least three times.The human gastric cancer cell line MGC-803 and human normal gastric epithelial cell line GES-1 were cultured in DMEM containing penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% FBS at 37 °C with 5% CO4/well and cultured at 37 °C with 5% CO2 for 24 h. And then, compounds (1–5) in various concentrations were introduced with incubation for another 48 h. Finally, 10 μL MTT (5 mg/mL) was added to each well. After 4 h incubation, the medium was removed and 150 μL DMSO was added. Absorbance was read on a Microplate Reader at 570 nm   in MGC-803 cells was examined using the Annexin V-FITC/PI detection kit, according to the description of previously reported  for 24 h. Cells were harvested and washed twice with cold PBS before incubating with annexin V and PI for 30 min in dark. And then the stained cells were detected and analyzed on FACS Calibur flow cytometry.The apoptosis effect of 2′-Z auraptene A  for 24 h and then harvested to detect the protein concentration using the BCA method. Total cellular protein was separated by an SDS/PAGE gel and transferred onto PVDF membranes using a semidry electroblotter. The membrane was blocked with non-fat milk solution (5%) for 1 h, and incubated subsequently with primary antibody at 4 °C overnight. Then TBST was used to wash the primary antibody and incubated with secondary antibody for 1 h at room temperature. Protein bands were visualized by enhanced chemiluminescence (ECL) and β-actin was used as a normalizing control.The detection of levels of apotosis-related proteins in MGC-803 cells was performed according to the reported western blot method  was obtained as colorless solid with a molecular formula of C16H18O4 determined from (+) HR-ESI–MS m/z 297.1094 . The NMR data of compound 2 , 4.60  and 5.51  indicated the presence of the partial structure of C-1′-C-2-C3′-C-7′, which was same with that of 1. Besides, considering the coupling relationships of the remaining three carbons, we confirmed the existence of the partial structure of C-3′-C-4′-C-5′. Therefore, we identified compound 2 as (Z)-7- oxy)- 2H-chromen-2-one and named it as 2′-Z auraptene A.2′-Z auraptene A (3) possessed a molecular formula of C19H22O4 determined from 13C-APT NMR data and (+) HR-ESI–MS ion peak at m/z 337.1406 . The NMR data of 3  in 1 were replaced by the primary oxygenated methylene signals . The substituted position of the primary oxygenated methylene in 3 was determined by the HMBC correlations from H-9′ to C-6′ and C-8′  and H-8′(δH 1.67) confirmed the configuration of C-6′ was Z. Finally, we established the new compound as 7--9′-hydroxy-3′,7′-dimethylocta-2′,6′-dien-1′-yl)oxy)-2H-chromen-2- one and we named it as 2′-Z auraptene B.2′-Z auraptene B (4) was isolated as colorless solid. Its molecular formula was deduced as C19H22O5 on the basis of 13C-APT NMR data and (+) HRESIMS ion peak at m/z 353.1355 . The NMR data of 4  and H-8′ (δH 1.70) were also observed in compound 4. Overall, we identified the new compound as 7--4′,9′-hydroxy-3′,7′-dimethylocta-2′,6′-dien-1′-yl)oxy)-2H-chromen-2-one and we named it as 2′-Z auraptene C.2′-Z auraptene C (5) was obtained as colorless solid. The molecular formula was established from 13C-APT NMR data and (+) HRESIMS ion peak at m/z 353.1356 . The NMR data of 4 almostly same with those of 5 (Tables c 142.80) shifted to high field region (δc 138.70) and C-6′ (δc 120.80) shifted to down field region (δc 127.30) reversely. Considering the significantly influence of the substituted position of C-4′ and C-5′ to C-3′ and C-6′, the position of the hydroxyl in 4 must be different from that of compound 5. In the HMBC spectrum, the correlations from H-5′ (δH 4.62) to C-3′ (δc 138.70) and C-8′ (δc 14.20)  and H-8′(δH 1.73) in the NOESY spectrum. Therefore, we determined the new compound as 7--5′,9′-hydroxy-3′,7′-dimethylocta-2′,6′-dien-1′-yl)oxy)-2H-chromen-2-one and we named it as 2′-Z auraptene D.2′-Z auraptene D , was formed through the hydroxylation of C-9′, which was further hydroxylated at C-4′ or C-5′ to obtain compound 4 or compound 5, respectively  and four new metabolites (2–5) obtained from the biotransformation of 1 by Mucor polymorphosporus at human gastric cancer MGC-803 cells and human epithelial cell line GES-1, as shown in Fig. 2 showed preferential pharmacological activities with an IC50 value of 0.78 ± 0.13 μM and therapeutic index (TI) value of 59.0, which are significantly improved from that of 2′-Z auraptene , which indicated that 2′-Z auraptene A (2) was the most potent against MGC-803 gastric cancer cells and less cytotoxic against GES-1 cell line compared with MGC-803 cells  on inducing apoptosis, MGC-803 cells were treated with compound 2 for 24 h and submitted to flow cytometry analyses. The celluar apoptotic rates in compound 2-treated groups increased significantly in a dose-dependent manner compared with the control group Below is the link to the electronic supplementary material.


The structures are reported of six racemic tris(bipyridyl)iron salts with a range of 2-substituted-1,1,3,3-tetracyanopropenide anions, mostly also containing either perchlorate or tetrafluoridoborate as co-anions. In three of the compounds the polynitrile anions are fully ordered, and in three others they are disordered, while the co-anion is also ordered in three compounds, but disordered in two others. Supramolecular assemblies range from no continuous aggregation up to a three-dimensional hydrogen-bonded framework structure. 10H8N2)3](C8H3N4O)2.0.776H2O, (I), trisiron(II) 1,1,3,3-tetracyano-2-(propylsulfanyl)propenide perchlorate, [Fe(C10H8N2)3](C10H7N4S)(ClO4), (II), trisiron(II) 1,1,3,3-tetracyano-2-methoxypropenide tetrafluoridoborate ethanol 0.926-solvate, [Fe(C12H12N2)3](C8H3N4O)(BF4).0.926C2H2O, (III), trisiron(II) 1,1,3,3-tetracyano-2-ethoxypropenide tetrafluoridoborate, [Fe(C12H12N2)3](C9H5N4O)(BF4), (IV), trisiron(II) 1,1,3,3-tetracyano-2-(ethylsufanyl)propenide tetrafluoridoborate, [Fe(C12H12N2)3](C9H5N4S)(BF4), (V), and trisiron(II) 1,1,3,3-tetracyano-2-propoxypropenide tetrafluoridoborate, [Fe(C12H12N2)3](C10H7N4O)(BF4), (VI). In compound (I), one of the anions is disordered over two sets of atomic sites with equal occupancies while, in the second anion, just one of the C(CN)2 units is disordered, again over two sets of atomic sites with equal occupancies: the anionic components are linked by multiple C—H⋯N hydrogen bonds to form a three-dimensional framework. In compound (II), the polynitrile anion is disordered over two sets of atomic sites with occupancies in the approximate ratio 3:1, while the perchlorate anion is disordered over three sets of atomic sites: there are C—N⋯π interactions between the cations and the polynitrile anion. The polynitrile anion in compound (III) is fully ordered, but the tetrafluoridoborate anion is disordered over two sets of atomic sites with occupancies 0.671 (4) and 0.329 (4): the cations and the tetrafluoridoborate anions are linked by C—H⋯F interactions to form an interrupted chain. Compounds (IV) and (V) are isostructural and all of the ionic components are fully ordered in both of them: the cations and tetrafluoridoborate anions are linked into C22(12) chains. The polynitrile anion in compound (VI) is disordered over two sets of atomic sites with approximately equal occupancies, and here the chains formed by the cations and the tetrafluoridoborate anions are of the C22(13) type.Structures are reported for six closely related salts of tris(bipyridyl)iron(II) cations, namely trisiron(II) bis 0.776-hydrate, [Fe(C The compounds were all prepared using solvothermal reactions between mixtures of iron(II) salts, a 2,2′-bipyridine and polynitrile salts of the type K(tcnX), where the substituent X is as defined above.The polynitrile anions all have the constitution 1,1,3,3-tetracyano-2-Compounds (I)–(VI) all contain a tris(bipyridine)iron(II) cation and a 2-substituted-1,1,3,3-tetracyanopropenide anion. In compounds (I)2 units over two orientations with occupancies which refined to values which are equal within experimental uncertainly, 0.501 (7) and 0.499 (7), while the other anion, containing atom O821, exhibits whole anion disorder, again over two sets of atomic sites with refined occupancies 0.502 (2) and 0.498 (2): all of these occupancies were therefore set to 0.5. In compound (II)In several of the compounds, the anions exhibit disorder. One of the propenide anions in compound (I)The propenide anion of compound (III)X −, readily acts as a ligand towards iron(II) –(VI) do the polynitrile units act as ligands towards the iron(II) centres, but they are always present as free anions. This is consistent with the behaviour observed in a wide range of other iron(II) complexes containing polypyridyl ligands as anions of the general type tcn2) units in each orientation is involved, but in (II)2) units in both orientations are involved in hydrogen bonds, thus tethering these anions at both ends. Hence, no plausible explanation of polynitrile order versus disorder can be gleaned from hydrogen bonding: nor do the C—N⋯π contacts provide any explanation, as there are more of these in (II)It is interesting to note that the polynitrile anions in compounds (II)–(V) are fully ordered while those in compounds (I)II; in comparable high-spin complexes, the Fe—N distances are always around 2.15 Å –(VI) all lie within a narrow range of less than 0.03 Å, with extreme values of 1.9579 (12) Å in (V)With the exception of the isostructural pair of compounds (IV)et al., 1994The supramolecular aggregation in compounds (I)–(VI) depends upon hydrogen bonds of a number of different types Table 1; nearly et al., 1998abet al., 20001 screw axis along on Fig. 7, while son Fig. 8. The comon Fig. 8. DespiteD—H⋯A angle greater than 140°, so that the others can probably be discounted as structurally unimportant et al., 1995The partial-occupancy ethanol component in compound (III)1 screw axis along  Fig. 11: the tcnn Fig. 12: again tet al., 2008et al., 2008−⋯centroid distances cluster around 3.1 Å, and F⋯centroid distances lie in the range 2.7–2.9 Å. Although no systematic studies have been made on N-containing anions, it is probable that optimal N⋯centroid distances in such systems will be less than the covalent C—Cl⋯centroid optimum distance of 3.6 Å. Thus, in the tris(phenethroline)iron(II) salt with the anion (tcnX)− where X here represents the 2-hydoxyethoxy group , one of the cyano groups forms contacts with two different pyridyl rings within the selected asymmetric unit, with N⋯centroid distances of 3.212 (2) and 3.418 (2) Å  of less than 3.4 Å The interactions between aromatic rings and both covalent C—Cl bonds and chloride ions have recently been reviewed –(VI) reported here, the bond distances in the anions indicate delocalization of the negative charge over the whole of the tetracyanopropenide skeleton of the anion.The structures of compounds containing tcnv/v, 20 cm3) were heated at 423 K for 3 d in a sealed Teflon-lined stainless steel vessel under autogenous pressure and then cooled gradually to room temperature at a rate of 10 K h−1. After the reaction vessels had cooled to ambient temperature, crystals suitable for single-crystal X-ray diffraction were collected by filtration and dried in air. For the synthesis of compounds (I)All chemical reagents and solvents are commercially available and were used without further purification. For the synthesis of compounds (III)–(VI), mixtures of 5,5′-dimethyl-2,2′-bipyridine , iron(II) tetrafluoridoborate hexahydrate , and 0.2 mmol of the appropriate polynitrile salt: .Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018015426/sj5565sup1.cifCrystal structure: contains datablock(s) global, I, II, III, IV, V, VI. DOI: 10.1107/S2056989018015426/sj5565Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989018015426/sj5565IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989018015426/sj5565IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989018015426/sj5565IVsup5.hklStructure factors: contains datablock(s) IV. DOI: 10.1107/S2056989018015426/sj5565Vsup6.hklStructure factors: contains datablock(s) V. DOI: 10.1107/S2056989018015426/sj5565VIsup7.hklStructure factors: contains datablock(s) VI. DOI: 1876478, 1876479, 1876480, 1876481, 1876482, 1876483CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


IDH1) occur at high frequency in grade II–III gliomas (LGGs). IDH1 mutations are somatic, missense and heterozygous affecting codon 132 in the catalytic pocket of the enzyme. In LGG, most mutations (90%) result in an arginine to histidine substitution (IDH1R132H) providing a neo-epitope that is expressed in all tumor cells. To assess the immunogenic nature of this epitope, and its potential use to develop T cell treatments, we measured IDH1R132H-specific B and T cell reactivity in blood and tumor tissue of LGG patients.Mutations in the isocitrate dehydrogenase-1 gene (R132H-mutated LGG patients (n = 27) were assayed for the presence of a neo-specific antibody response using ELISA. In addition, PBMCs (n = 36) and tumor-infiltrating lymphocytes  were measured for T cell activation markers and IFN-γ production by flow cytometry and ELISA. In some assays, frequencies of CD4 T cells specific for mutated peptide presented by HLA-DR were enriched prior to T cell monitoring assays.Sera from IDH1R132H-specific IgG in sera of LGG patients. Similarly, we did not observe CD4 T cell reactivity towards IDH1R132H in blood, neither did we observe such reactivity following pre-enrichment of frequencies of IDH1R132H-specific CD4 T cells. Finally, we did not detect IDH1R132H-specific CD4 T cells among TILs.Despite high sensitivity of our assay, we failed to detect IDH1R132H is not sufficiently immunogenic and devaluates its further therapeutic exploitation, at least in the majority of LGG patients.The absence of both humoral and cellular responses in blood and tumors of LGG patients indicates that IDH1The online version of this article (10.1007/s11060-019-03228-6) contains supplementary material, which is available to authorized users. IDH) 1 or 2 genes and are classified as diffuse low-grade gliomas (LGG). Grade IV glioma are classified as high-grade glioma (HGG) and can be distinguished in either primary (IDH wildtype) or secondary (IDH mutant) gliomas  was used as positive control, to which end, rabbit anti-mouse-HRP antibody ) was added as second step. After a final wash step, 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich) was added and incubated until color developed. Reaction was stopped by addition of 1 M hydrochloric acid, and optical densities (OD) at 450 nm were measured using a Multiskan Ascent Plate Reader (ThermoFisher). All experiments were performed at room temperature.Pierce Streptavidin Coated High Sensitivity Plates  were washed in a HydroFlex microplate washer  using PBS/0.05% Tween20 (Sigma-Aldrich). Plates were coated with 3 µM peptide in PBS/0.05% Tween20 for 1 h. Plates were washed and PBS/0.05% Tween20 + 10% FBS was added to each well (1 h) in order to reduce non-specific antibody binding. Sera from IDH16 cells) were stimulated with 17.5 µM IDH1R132H, IDH1wt, MOG, or 1 µg/mL staphylococcus-derived enterotoxin B  in 400 µL Iscove’s Modified Dulbecco’s Medium (IMDM) containing 2% l-glutamine, 1% penicillin and streptomycin, supplemented with 6% allogeneic human serum at 37 °C for 16 h.PBMC were isolated from heparin blood by density-gradient centrifugation using Ficoll-Paque PLUS (Sigma-Aldrich). Viable cells and leukocytes were stained using Tuerk solution (Sigma-Aldrich) and Tryptan Blue (ThermoFisher), and microscopically counted. Patient PBMC (2 × 105/well) together with autologous patient PBMC (0.4 × 105/well) and 17.5 µM or no peptide in IMDM supplemented with 10 ng/mL IL-7 (200 µL/well). Cells were co-cultured for 3–4 weeks and additional cytokines were added. Day 0: 10 ng/mL IL-7, day 3: 22 ng/mL IL-15, day 6: 100 IU/mL IL-2, day 13: 10 ng/mL IL-7, 1 ng/mL IL-15 and 100 IU/mL IL-2. Cells were split using IMDM containing 360 IU/mL IL-2. Following co-culture, T cells were harvested, washed and assayed (0.2 × 106) for their reactivity upon stimulation with peptide-loaded DR1+, DR4+ or non-transduced HeLa cells (0.2 × 106) at 37 °C for 16 h.In some experiments, effector T cells were co-cultured together with peptide-loaded antigen presenting cells prior to peptide stimulation. HeLa cells stably expressing DRA1*01:01 and DRB1*01:01 (DR1 + HeLa) or stably expressing DRA1*01:01 and DRB1*04:01 (DR4 + HeLa)   were irrl-glutamine, 25 mM HEPES, 1% penicillin and streptomycin, supplemented with 6% allogeneic human serum and 1000 IU/mL IL-2 (TIL medium) at 37 °C. After 2 weeks of culture, contents of wells with clearly visible lymphocyte growth were pooled for each LGG. Viable cells and leukocytes were stained using Tuerk solution (Sigma-Aldrich) and Tryptan Blue (ThermoFisher), and subsequently microscopically counted. Successful cultures of TILs were verified with CD3 flow cytometry (see “6) were plated with 0.5 × 106 target cells  or 1 µg/mL SEB in 200 µL IMDM containing 2% l-glutamine, 1% penicillin and streptomycin, supplemented with 6% allogeneic human serum at 37 °C for 16 h.TIL microcultures were initiated and expanded from tumor fragments as described previously . In shortry see “” below. Following T cell stimulations, 2.5 µL PerCP-conjugated mouse anti-human CD45 , 5 µL FITC-conjugated mouse anti-human CD3 (BD Biosciences), 5 µL PE-conjugated mouse anti-human CD4  and 5 µL APC-conjugated mouse anti-human CD137 (BD Biosciences) were premixed in 50 µL PBS and added to cell pellets. Samples were incubated at 4 °C for 30 min, fixed with paraformaldehyde 1% and measured on a FACS Canto (BD Biosciences). Lymphocyte populations were gated using forward scatter (FSC) and side scatter (SSC) plots, and within these lymphocytes CD45+ cells and CD4+ T cells were sequentially gated, after which the latter cells were assessed for CD137 surface expression . Flow cytometric analysis was performed using FCS Express 4 Flow software. Supernatants of T cells following stimulation with peptides or controls were analyzed for the presence of IFN-γ using Human IFN-γ ELISA Ready-SET-Go! kit (ThermoFisher) according to the manufacturer’s instructions.Statistical analysis was performed using RStudio software and statistical methods used are specified in the figure legends.R132H-specific IgG using sera from LGG patients who harbored the IDH1 mutation (n = 27). Healthy donor sera were included as negative controls (n = 5). Using the experimental setup as depicted in Fig. R132H peptide  and were subsequently stimulated with IDH1R132H peptide loaded, irradiated autologous PBMCs. Also using TILs, we were not able to observe changes with respect to CD137 expression and IFN-γ production of CD4 T cells for 10 out of 10 patients , PBMC (n = 30) as well as TILs (n = 10), we showed that immune cell populations with reactivity towards mutant IDH1 are not present in the LGG patient samples studied here.In this study, we focused on detecting the presence of IDH1These findings are in contrast to previous observations where Schumacher and colleagues demonstrated humoral and CD4 T cell responses in patients with glioma . In our R132H-specific antibodies or frequencies of IDH1R132H-specific CD4 T cells may be below the detection thresholds of assays. For instance, among TILs from (multiple and pooled) IDH1R132H-mutant tumors, the frequency of IDH1R132H-specific CD4 T cells was less than 2% (of CD4+ T cells) after vaccinating mice with mutant peptides [d-2-hydroxyglutarate has also recently been described in LGG [R132H-specific CD4 T cells. A tentative low frequency of IDH1R132H-specific CD4 T cells was addressed by pre-enrichment of these cells by co-culturing PBMC of IDH1R132H-mutant patients with HeLa cells presenting IDH1R132H. None of the samples showed a IDH1R132H-specific T cell activation response, whether or not CD4 T cells underwent co-culture cycles with IDH1R132H-presenting cells [R132H-specific T cells cannot be dismissed, and these could be targeted with enrichment protocols using more professional antigen-presenting cells, optimal pMHC bindings [R132H [R132H is low.First, levels of IDH1peptides . Indeed,peptides . Along td in LGG , which mng cells , 43. Altbindings  or in vis [R132H , our datR132H, there is > 99% or 91% chance of detecting such responses in population frequencies of at least 10% or 5% respectively.Second, the number of patients assayed may have been too small in order to detect a cellular  or humoral immune response (n = 27). Percentages of patients with antibody or CD4 T cell responses in blood have been reported to be as low as 10% and 16%, respectively . HoweverR132H may be presented by specific HLA alleles that are underrepresented in our cohort of tested patients and/or that expression levels of IDH1 are too low to elicit IDH1R132H-specific T-cell responses. Indeed, glioblastomas express IDH1 at higher levels compared to gliomas of lower grade , grade III (n = 85) and grade IV gliomas (n = 159) respectively [Third, we cannot exclude that IDH1ectively ). HoweveR132H in blood or tumors of LGG patients. We advocate further preclinical studies prior to development and clinical exploitation of T cell treatments directed against IDH1R132H in LGG patients.Taken together, our data does not provide evidence for detectable presence of immune cell reactivity towards IDH1Supplementary file1 ; n.d., not determined. Supplementary table 2. Peptides used in functional assays. Supplementary fig. 1. Gating strategy to assess frequency of IDH1R132H-specific CD4+ T cells. Using flow cytometry, viable lymphocyte populations were first gated on FSC/SSC, after which CD45+ cells were gated and CD137 surface expression was assessed within CD4+ T cells.Below is the link to the electronic supplementary material.


V. vinifera grape varieties. In general, these compounds are studied for grape chemotaxonomy and for nutraceutical purposes due to their relevant antioxidant activity. Non-anthocyanic flavonoid composition of five red hybrid grape varieties produced by crossing of V. vinifera, V. aestivalis, V. cinerea, V. berlandieri, V. labrusca, V. lincecumii, and V. rupestris were studied by liquid chromatography/high-resolution mass spectrometry. Thirty-one compounds were identified, including methylnaringenin, a tetrahydroxy-dimethoxyflavanone-hexoside, two flavonols (quercetin and a pentahydroxyflavone isomer), 20 glycoside flavonols , three flavan-3-ols (−)-epicatechin, (+)-catechin, (−)-epicatechin gallate) and four proantocyanidins . Seibel 19881, Seyve Villard 12-347 and Seyve Villard 29-399 were particularly rich in polyphenols. These findings emphasize that these grapes are especially interesting for the production of antioxidant extracts for nutraceutical and pharmaceutical uses.Previous studies showed that hybrid grapes often have qualitatively and quantitatively higher polyphenolic contents than the common  French paradox − ion isotopic pattern and confirmed by MS/MS analysis. In the five grape samples, thirty-one non-anthocyanic flavonoids reported in O-rhamnoside, kaempferide-p-coumaroylhexoside, and isorhamnetin-glucuronide—had a lower identification score  probably due to their low signal intensity. For glucoside and glucuronide flavonols different characteristic fragmentations, were observed: glucoside compounds showed high signal of radical aglycone ion [Y0 − H]−• instead the glucuronide derivatives had higher signal of aglycone ion Y0−. A putative quercetin isomer was also identified: main MS/MS fragments at m/z 149.0244, which corresponds to the formula C8H5O3, and at m/z 151.0035 of ion C7H3O4, were observed  of compounds relies on the measurement of the mono-isotopic mass and isotopic pattern  it was possible to estimate the semi-quantitative content of the compounds reported in in grape . They weO-glucoside between 12.4 and 41.5 mg/kg) and the highest flavanol was procyanidin B1 (between 1.5 and 9.7 mg/kg). Similarly, high contents of procyanidin B1 and myricetin-3-O-glucoside were found in the V. labrusca grape variety Bordô and in skin of the table grape varieties BRS Clara and BRS Morena  × (Aramon × Ganzin)} × [(V. lincecumii × V. rupestris) × Aramon]; Seibel 5455 = {[(Aramon × Ganzin) × Bouboulenc (V. vinifera)] × [(V. lincecumii × V. rupestris) × ] × V. berlandieri}; VIVC number 2768, http://www.vivc.de/), Seibel 19881 , Seyve Villard 29-399  and Seyve Villard 12-347 (Seibel 6468 × Seibel 6905: Seibel 6468 = {[(Aramon × Ganzin) × Bouboulenc] × Sicilien (V. vinifera)]} × {[(V. lincecumii × V. rupestris) × ] × V. berlandieri} × [(V. vinifera × V. labrusca) × V. rupestris] × V. vinifera; Seibel 6905 = Seibel 451  × {[(V. lincecumii × V. rupestris) × V. vinifera] × (Aramon × Ganzin)} × {[(V. lincecumii × V. rupestris) × V. vinifera] × (Aramon × Ganzin)} × {[(V. rupestris) × V. rupestris)] × Blanc Royal}; VIVC number 11587). Grapes were harvested in 2011 and 2012 at ripeness from the five plants of each variety present in the CREA-VIT grapevine germplasm repository . Mean sugar content of samples at the harvests were: Unknown hybrid red grape variety °Brix = 18.2 ± 0.8; Seibel 8357 °Brix= 19.4 ± 1.0; Seibel 19881 °Brix = 21.3 ± 1.1; Seyve Villard 29-399 °Brix = 17.7 ± 0.2; Seyve Villard 12-347 °Brix = 18.9 ± 1.0. For each sample, about 100 berries were picked randomly and immediately frozen at −20 °C. For sample preparation, twenty berries were weighed and homogenized using liquid nitrogen and the resulting powder was immediately extracted with pure methanol in ratio 2:1 v/w under stirring for 20 min in the dark. After addition to the extract of 200 μg/L of a 4ʹ,5,7-trihydroxyflavanone 500 mg/L solution as internal standard, the vial sample was wrapped with an aluminum foil to limit possible photo-oxidation and centrifuged at 10 °C for 20 min. The solution was filtered with an Acrodisc GHP 0.22 μm filter  and collected in a vial for LC/MS analysis. To perform quantitative analysis a standard solution was analyzed and RFIS/RFanalyte ratio of each compound was calculated. RFIS/RFanalyte coefficients were: Procyanidin B1 = 29.2, procyanidin B2 = 58.4; quercetin = 2.4; quercetin-3-O-glucoside = 8.2; rutin = 12.7; isorhamnetin-3-O-glucoside = 2.4; kaempferol-3-O-glucoside = 2.5; myricetin-3-O-glucoside = 14.1; (+)-catechin = 5.8; (−)-epicatechin = 6.6; (−)-epicatechin gallate = 16.7; (−)-epigallocatechin gallate = 64.3; (−)-epigallocatechin = 11.1.Standards of myricetin-3-v/v aqueous formic acid; and (B) 0.1% v/v formic acid in acetonitrile. Gradient elution program: 5% B isocratic for 8 min, from 5% to 45% B in 10 min, from 45% to 65% B in 5 min, from 65% to 90% in 4 min, and 90% B isocratic for 10 min. Flow rate was 0.4 mL/min; sample injection was 10 μL; column temperature was 35 °C.The analytical system used was Agilent UHPLC 1290 Infinity coupled to Agilent 1290 Infinity Autosampler (G4226A) and Agilent 6540 accurate-mass Q-TOF mass spectrometer  with Jet Stream Ionization source . For each sample, two negative ionization mode analyses were performed with full scan acquisition. After each sample a blank was run. The data acquisition software was Agilent MassHunter version B.04.00 (B4033.2). Chromatography was performed using a Zorbax reverse-phase column  (Agilent Technologies). The mobile phase was composed of (A) 0.1% QTOF conditions: sheath gas nitrogen 10 L/min at 400 °C, drying gas 8 L/min at 350 °C, nebulizer pressure 60 psi, nozzle voltage 1 kV, and capillary voltage 3.5 kV. Signals in the m/z 100–1700 range were recorded. Negative mass calibration was performed with standard mix G1969-85000  and had residual error for the expected masses between ±0.2 ppm. Lock masses were: TFA anion at m/z 112.9856 and HP-0921(+formate) at m/z 966.0007 in negative-ion mode.MS/MS conditions: collision energies between 20 and 60 eV were used to fragment ions in the m/z 100–1700 range. Acquisition rate was 2 spectra/s.Data processing was performed with Agilent Mass Hunter Qualitative Analysis software version B.05.00 (5.0.519.0) (Agilent Technologies). Confidence of the compound identification was based on accurate mass and isotope pattern and was expressed by an “overall identification score” computed as a weighted average of the compound isotopic pattern signals, such as exact masses, relative abundances, and m/z distances (spacing). Weight parameters were Wmass = 100, Wabundance = 60, and Wspacing = 50; mass expected data variation 2.0 mDa + 5.6 ppm, mass isotope abundance 7.5%, and mass isotope grouping peak spacing tolerance 0.0025 m/z + 7.0 ppm.Among the samples studied, Seibel 19881 and the two Seyve Villard varieties are characterized by peculiar richness of non-anthocyanic flavonoids. In particular, Seyve Villard samples have high content of flavan-3-ols, procyanidin B1 and procyanidin B2. It is worth to highlight that these compounds have protective activity towards LDL oxidation greater than standard antioxidants like trolox and ascorbic acid . Seibel A previous study showed that these Seyve Villard grape varieties have low anthocyanin content . On the