II complexes of 1-H-3,4,5-trimethylpyrazole, cis-2}2(μ-Cl)2] and (NO3)2, are presented and compared to the 3-methyl-1H-pyrazole and 3,5-dimethyl-1H-pyrozole analogues.The crystal structures of two Cu H-pyrazole with CuCl2·2H2O and Cu(NO3)2·2.5H2O are presented, namely di-μ-chlorido-bis[chloridobiscopper(II)], [Cu2Cl4(C6H10N2)4] (1) and aquatetrakiscopper(II) dinitrate, [Cu(C6H10N2)4(H2O)](NO3)2 (2), and compared to the previously determined structures for 3-methyl-1H-pyrazole and 3,5-dimethyl-1H-pyrazole. CuCl2 forms a 2:1 ligand-to-metal chloride-bridged complex with 3,4,5-trimethyl-1H-pyrazole, with a square-pyramidal coordination geometry about each copper(II) center. Similarly to the previously obtained 3,5-dimethyl-1H-pyrazole complex with CuCl2, the pyrazole ligands are cis to each other, with two chloride ions bridging the two copper(II) centers, and a terminal chloride ion occupying the axial position. Cu(NO3)2 forms a 4:1 ligand-to-metal complex with 3,4,5-trimethyl-1H-pyrazole that is also arranged in a square-pyramidal geometry about CuII. The newly obtained copper(II) complex has the same coordination geometry as the 3,5-dimethyl-1H-pyrazole complex, including an axial water molecule, two nitrate ions hydrogen-bonded to the water molecule, and four pyrazole ligands in the equatorial plane, suggesting that similar steric forces are at play in the formation of these complexes.The crystal structure of complexes of 3,4,5-trimethyl-1 II complexes from water 2 were used to assess counter-ion effects on the crystal structure in a manner consistent with the previous work.Pyrazoles are a useful class of molecules because they coordinate with metal ions, form conjugated π-systems, and can be tuned electronically and sterically through a number of possible substituent groups. It is therefore important to gain a better understanding of how changes in reaction conditions, including solvent, substituents, and counter-ions, affect the structures of compounds incorporating pyrazole and its derivatives. Previous work using mono- and dimethyl pyrazole ligands demonstrated the effect of the counter-ion on the final structure and electronic properties of their respective Cu2 complex with 1-H-3,4,5-trimethylpyrazole , which is related to the first CuII by an inversion center. The overall geometry around each CuII center is square pyramidal, with the axial position occupied by the elongated bridging chloride contact, and the equatorial positions occupied by the two trimethylpyrazole ligands in a cis configuration, one terminal chloride ion, and the shorter bridging chloride contact. In 1, the trimethylpyrazole ligands are tilted off-perpendicular from the basal plane of the square-pyramidal CuII coordination environment. The dihedral angles of the pyrazole ligands to the basal plane and to each other are as follows: between the mean N9/N10/C11–C13 plane and the mean Cl2/Cl1/N2/N10 plane, 53.9 (2)°; between the mean N2/N1/C3–C5 plane and the mean Cl2/Cl1/N2/N10 plane, 47.1 (2)°; between the mean N9/N10/C11–C13 and the mean N2/N1/C3–C5 plane, 51.5 (2)°. The trimethylpyrazole ligand is not deprotonated in the complex as there are two chloride ions per CuII ion. Additionally, the bond distances within the trimethylpyrazole ring are more characteristic of a non-aromatic, conjugated ring , rather than the heteroaromatic species obtained upon deprotonation 2 and 1-H-3,4,5-trimethylpyrazole, the structure consists of a single CuII center exhibiting a square-pyramidal coordination geometry, oriented such that the pyrazole ligands occupy the four planar positions around CuII, with a water molecule occupying the axial position. The pyrazole ligands are oriented so that the non-coordinated pyrazole nitrogen atoms are cis across the N—Cu—N bonds on opposite sides of the structure, and are trans across the N—Cu—N bonds on adjacent pyrazoles. Additionally, in 2 as in 1, the trimethylpyrazole ligands are tilted off-perpendicular from the basal plane of the square-pyramidal CuII coordination environment. The dihedral angles of the pyrazole ligands to the basal plane and to each other are as follows: between the mean N9/N10/C11–C13 and the mean N2/N10/N10*/N2* plane, 49.2 (2)°; between the mean N2/N1/C3–C5 plane and the mean N2/N10/N10*/N2* plane, 61.3 (2)°; between the mean N9/N10/C11—C13 and the mean N2/N1/C3–C5 plane, 78.7 (2)° . The nitrate ions are not directly coordinated to CuII. This structure is similar to that obtained from 1-H-3,5-dimethylpyrazole and Cu(NO3)2 in water, which reinforces the conclusion that steric effects likely play a part in determining how the ligands orient themselves in this complex –1.405 (4) Å; C4—C5, C12—C13, 1.379 (4) and 1.373 (4) Å, respectively; C13—N9, 1.341 (3) Å; C11—N10, 1.336 (3) Å, C3—N2, 1.332 (3) Å; C5—N1, 1.349 (4) Å; N9—N10, 1.369 (3) Å; N1—N2, 1.358 (3) Å] providing further evidence for a neutral trimethylpyrazole ligand  and very weak intermolecular interactions involving the same Cl2 atoms and N—H groups of adjacent complexes  Å between CuII centers of complexes within stacked columns] when compared to the dimethylpyrazole . Both structures crystallize in space group I2/a (reported as C2/c for the dimethylpyrazole complex).In s Table 2. Surpriser Fig. 4 than itset al., 2016H-3,4,5-trimethylpyrazole yields only 17 entries. The structure with CSD refcode FITQEE is of the neutral ligand only  center , C—N (1.308–1.365 Å), and N—N (1.338–1.374 Å) bond distances within the pyrazole group are similar to those in 1, but cover a wider range. Complexes structurally similar to 2 can be found in the CSD with refcodes FAYTOO , 1.366–1.389 Å (MIFYUW), and 1.369–1.400 Å (YUXSEP01); C—N bond length ranges of 1.270–1.433 Å (FAYTOO), 1.329–1.346 Å (MIFYUW), and 1.334–1.350 Å (YUXSEP01); and N—N bond length ranges of 1.322–1.477 Å (FAYTOO), 1.361–1.375 Å (MIFYUW), and 1.360–1.365 Å (YUXSEP01).A complex structurally similar to 2·2H2O was purchased from Aldrich, and Cu(NO3)2·2.5H2O was purchased from Fisher. Deionized water was used in all reactions.All manipulations were carried out in air at room temperature with reagents as obtained from the manufacturer, unless otherwise stated. 3-Methyl-2,4-pentanedione was purchased from Alfa–Aesar, while the hydrazine monohydrate was purchased from Sigma–Aldrich. CuCl1-H-3,4,5-trimethylpyrazole: Following literature procedure 3(C3H2N2)]2}2(μ-Cl)2] (1):-(NO3)2 (2)[Cu{3,4,5- was dissolved in 5 mL of H2O, with 1 mL of acetone added to aid dissolution. A light-blue solution of 0.18296 g (0.78655 mmol) Cu(NO3)2·2.5 H2O in 5 mL H2O was added via pipette to this solution while stirring. A 1 mL rinse of the CuII vessel with H2O was added to the reaction. There was an immediate change of color to light green as the CuII solution was added, which darkened upon further addition of the CuII solution, reaching dark green. Upon complete addition of CuII, the solution became teal green with a small amount of precipitate. The reaction was stirred overnight, filtered, and the solvent slowly evaporated to yield dark-blue crystals.1, five reflections (110), (100), (010), (001), and (111) were omitted from the final refinement on account of beamstop truncation. For 2, five reflections (110), (200), (011),  = 1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018002359/vm2208sup1.cifCrystal structure: contains datablock(s) global, 1, 2. DOI: 10.1107/S2056989018002359/vm22081sup2.hklStructure factors: contains datablock(s) 1. DOI: Click here for additional data file.10.1107/S2056989018002359/vm22081sup4.cdxSupporting information file. DOI: 10.1107/S2056989018002359/vm22082sup3.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989018002359/vm22082sup5.cdxSupporting information file. DOI: 1823018, 1823017CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Double deficiency of p38α and p38β in naïve CD4+ T cells resulted in an attenuation of MAPK-activated protein kinase (MK)-dependent mTOR signaling after T cell receptor engagement, and enhanced their differentiation into regulatory T cells under appropriate inducing conditions. Pharmacological inhibition of the p38-MK-mTOR signaling module produced similar effects, revealing potential for therapeutic applications.The evolutionarily conserved protein kinase p38 mediates innate resistance to environmental stress and microbial infection. Four p38 isoforms exist in mammals and may have been co-opted for new roles in adaptive immunity. Murine T cells deficient in p38α, the ubiquitously expressed p38 isoform, showed no readily apparent cell-autonomous defects while expressing elevated amounts of another isoform, p38β. Mice with T cells simultaneously lacking p38α and p38β displayed lymphoid atrophy and elevated Foxp3 The T cell receptor (TCR)Homologues of p38, a MAPK family member, are found in organisms ranging from yeast to humans. In single-celled life, p38 is activated upon stress and damage, and signals to evoke cell-autonomous mechanisms for adaptation, repair, and survival , 2. The 3–9–in vivo effects of impaired p38 function in T cells by using pharmacological p38 inhibitors  cell induction and attenuated allergic inflammation when challenged with epicutaneous antigen. fl/fl-LckCreMapk14; Δα) showed moderate lymphoid atrophy, and had decreased proportions of CD8+ subsets among lymph node and splenic T cells , and further elevated in lymph node and splenic T cells with p38α ablation (B).We previously reported that mice with T cell-specific ablation of p38α  to generate mice with T cells lacking both p38α and p38β . T cells from Δαβ mice did not express detectable amounts of both p38 isoforms (C). The size and cellularity of the thymus, lymph nodes, and spleen of Δαβ mice decreased compared with those of wild-type (WT) mice (D and E). The lymphoid tissues of Δα and Δβ mice tended to be atrophic too, but to lesser extents than Δαβ counterparts. Single and double deficiency of p38α and p38β in T cells did not prevent the thymic development of CD4+ and CD8+ T cells or their homing to and maintenance in the lymph nodes and spleen (F). In keeping with our previous report .To examine whether p38α and p38β exert redundant functions in T cells, we crossed Δα mice with p38β-null mice (isoforms C. The siWT) mice , D and Ed spleen F. In kees report , the proΔαβ mice F.+ T cells. Compared with T cells lacking only either isoform, T cells from Δαβ mice exhibited a more complete abrogation of p38 phosphorylation after treatment with TCR agonists, anti-CD3 and anti-CD28 antibodies (A and B). Loss of p38 has been shown to potentiate signaling by other MAPK family members, ERK and JNK, in a multitude of cell types (A). We observed that MAPK-activated protein kinase (MK) 2, a protein kinase immediately downstream of p38, was constitutively phosphorylated in cultured T cells independently of TCR stimulation. Nevertheless, MK2 phosphorylation was abolished in Δαβ T cells, but not Δα and Δβ T cells (A and B), suggesting that p38 was indispensable for steady-state phosphorylation of MK2.We examined the effects of p38 deficiency on TCR-induced intracellular signaling in naïve CD4tibodies , A and Bll types . Correspll types A. We obs T cells , A and BA). In parallel, mTOR pathway activation, as indicated by S6K and S6 phosphorylation, was greatly weakened in Δαβ T cells but persisted in Δα and Δβ T cells, albeit in lower magnitudes than in WT counterparts (A and B). TCR-induced S6 phosphorylation was also diminished in CD4+ T cells lacking MK2 and the related protein kinase MK3 (A–E), which served overlapping signaling functions (+ T cells lacking MK2 and MK3 (F and G). In summary, p38-dependent activation and regulation of intracellular signaling events in naïve CD4+ T cells were abolished by ablation of both p38α and p38β but not either alone.Some previous studies suggested a connection between the p38-MK2 signaling module and the mechanistic target of rapamycin (mTOR): p38 was required for stress-induced mTOR signaling in arsenite-exposed fibroblasts, hypoxic-reoxygenated cardiomyocytes, and anisomycin-treated macrophages –37. Anis–Fig. 2nase MK3 , A–E, whunctions , 40. Int and MK3 , F and G+) splenic T cells from WT and αβΔ mice and stimulated them with anti-CD3 and anti-CD28 antibodies. The proliferation rate of TCR-stimulated CD4+ T cells was substantially reduced by p38α and p38β deficiency (A). These effects could indicate a cell-autonomous role for p38 signaling in TCR-induced proliferation or, alternatively, a contribution of a non-cell-autonomous mechanism that suppressed T cell activation more effectively without the p38 isoforms. Treg cells, contained in the total splenic T cell preparations, might exert the suppressive effect under the latter scenario.We sought to identify functional defects in T cells that resulted from loss of p38 signaling. First, we isolated total  compared with the effects seen with total splenic T cells. Naïve CD4+ T cells from WT and Δαβ mice produced comparable amounts of IFN-γ, IL-13, and IL-17A upon TCR stimulation (C), and differentiated into the effector cell subsets specialized for producing these cytokines  with similar efficiencies (D and E). Therefore, p38 signaling in T cells seemed largely dispensable for TCR-driven activation and differentiation toward Th effector cells.To exclude or minimize the possible influence of Treg cells, we performed subsequent experiments using cell preparations devoid of antigen-experienced T cells including Foxp3ed rates B comparemulation C, and diciencies , D and E+ T cells from Δαβ mice differentiated into Treg cells more efficiently than WT counterparts (A and B). Consistently, T cells from Δαβ mice produced greater amounts of Foxp3 mRNA and protein than WT cells during the course of Treg cell induction (C and D). TCR-responsive Foxp3 mRNA induction in the absence of a Treg-skewing condition was also higher in naïve CD4+ T cells from Δαβ mice (C), suggesting that p38 signaling served to limit Foxp3 expression even before the Treg phenotype was established. The suppressive function of Treg cells from Δαβ mice, as determined by the ability to inhibit TCR-induced proliferation of naïve CD4+ T cells, was intact, and in fact slightly enhanced compared with that of WT Treg cells (E).We examined whether p38 signaling played a role in the induction of Treg cells. Upon TCR stimulation in conjunction with exposure to TGF-β, naïve CD4terparts , A and Bnduction , C and DΔαβ mice C, suggeseg cells E.in vivo. Notably, Δαβ mice in a steady-state condition exhibited a small but consistent increase in Treg cell frequency: CD4+ T cells from the thymus and spleen of Δαβ mice contained elevated Foxp3+ fractions (F and G). To examine Treg cell induction during T cell-mediated immune responses, we subjected WT and Δαβ mice to an experimental condition that elicited contact hypersensitivity reactions. In this model of allergic dermatitis, epicutaneous exposure to a small-molecule antigen or hapten establishes T cell immunity and, upon re-encounter with the hapten, induces T cell-mediated inflammation accompanied by skin rashes and swelling. Δαβ mice displayed greatly attenuated responses to the hapten 2,4-dinitrofluorobenzene  and, simultaneously, a massive expansion of the Treg cell compartment in the skin-draining lymph node (I and J).We next investigated Treg cell formation ractions , F and Gne DNFB; H and, simph node , I and J+ and CD4+Foxp3+ T cell subpopulations. It was conceivable that these effects resulted from impaired p38 signaling in cell types other than T cells, as Δαβ mice had systemic p38β deficiency. To address this possibility, we examined the lymphoid tissues of RAG1-deficient mice that were sublethally irradiated and engrafted with bone marrow from Δαβ mice. These radiation chimeras, in which deficiency of not only p38α but also p38β was restricted to lymphocytes, exhibited decreased CD8+ and increased CD4+Foxp3+ T cell subsets compared with control chimeras engrafted with WT bone marrow (A and B). Hence, the changes in the frequency of T cell subsets in Δαβ mice were likely attributable to the loss of cell-autonomous p38 function. The influence of Cre recombinase-mediated toxicity, if any, was also ruled out based on the normal ranges of CD8+ and CD4+Foxp3+ T cell abundance in LckCre mice (C and D).Given the observations described above, p38 signaling was not essential for T cell development and steady-state maintenance. Nevertheless, p38 deficiency in T cells produced perceptible effects in Δαβ mice—altered sizes of CD8e marrow , A and BCre mice , C and D+ T cells: increased Foxp3 expression and enhanced induction of fully functional Treg cells. We sought to identify the molecular mechanism whereby p38 signaling modulated Treg cell induction, and explored the possible involvement of the protein kinases whose activation was regulated by or dependent on p38. Excessive activation of ERK and JNK, seen in TCR-stimulated Δαβ T cells (A and B), might contribute to enhancing Treg cell induction; we refuted this possibility, however, as pharmacological inhibition of their activation did not significantly reduce the efficiency of Treg cell induction from Δαβ T cells (A). Further, deficiency of mitogen- and stress-activated kinase (MSK) 1 and MSK2, which are phosphorylated by and relay signals from ERK and p38 (B).Our analysis of Δαβ mice revealed novel effects of loss of p38 signaling in CD4 T cells , A and B T cells A. Furthe and p38 , did not and p38 B.+ T cells upon TCR stimulation but substantially diminished in Δαβ and MK2/3-double knock-out (DKO) counterparts . We therefore explored MK2/3 and mTOR as possible links for p38-dependent modulation of Treg cell induction. Indeed, pharmacological inhibition and genetic ablation of mTOR have been shown to enhance Foxp3 expression in CD4+ T cells, and promote their differentiation into Treg cells (C and D). Further, these compounds augmented in vitro Treg cell induction to similar extents (E).As shown above, S6 protein phosphorylation, an event downstream of mTOR activation, was induced in WT CD4eg cells , 42, pareg cells , C and D extents E.+ and CD8+ T cells (A). We compared the relative fitness of WT and MK2/3-DKO T cells in Treg cell formation in vitro by mixing equal numbers of naïve CD4+ T cells from WT CD45.1+ mice and MK2/3-DKO CD45.2+ mice and subjecting them to a Treg-skewing condition. The contribution of CD45.2+ cells to the Treg cell pools obtained at day 5 was greater than that of CD45.1+ cells (B and C). The efficiency of naïve-to-Treg conversion was also significantly higher with MK2/3-DKO T cells (D). In addition, similar to Δαβ mice, MK2/3-DKO mice exhibited a subtle but significant increase in steady-state Treg cell frequency in the spleen (E and F). In summary, our findings point to the p38α/β-MK2/3-mTOR cascade as a new actionable target for promoting the induction of immunosuppressive Treg cells.To obtain genetic evidence for MK2/3-dependent regulation of Treg cell induction, we investigated T cells isolated from MK2/3-DKO mice. Double deficiency of MK2 and MK3 in mice did not perturb the development and maintenance of CD4 T cells A. We com1+ cells , B and C T cells D. In adde spleen , E and Fex vivo for adoptive cell transfer therapy.A role for the p38 signaling pathway in immune responses was suspected early on, and the promise of effectively treating immune-mediated diseases by suppressing this pathway has been driving efforts to understand how this pathway contributes to immunity and inflammation. The findings of this study suggest a role for p38 signaling in several aspects of T cell biology: mice with T cell-restricted ablation of p38α and p38β expression exhibited reduced numbers of T cells and increased frequencies of Treg cells in lymphoid tissues. The decreases in contact hypersensitivity reactions in these mice were likely attributable to either or both of these two effects. From an extensive investigation of protein kinases activated downstream of the TCR, we identified the p38α/β-MK2/3-mTOR cascade as a key signaling module regulating Treg cell induction while ruling out the involvement of ERK, JNK, and MSK. Pharmacological agents interfering with this multilayered protein kinase module may improve the efficiency of generating or expanding Treg cells + T cells and stabilize it in Treg-committed cells. Cytokines provide major cues for the skewing of CD4+ T cell differentiation, but the strength of TCR signaling also contributes to determining the fate of activated T cells and, in particular, the efficiency of Treg cell formation , Δβ (Mapk11−/−), and MAPK-activated protein kinase (MK) 2/3-double knock-out (DKO) mice were previously described (fl/fl-LckCre-Mapk11Mapk14−/−) mice were generated by crossing Δα and Δβ mice. All mice were on a C57BL/6 background. Contact hypersensitivity to DNFB was induced as described (Rag1−/− mice (The Jackson Laboratory) were sublethally irradiated (450 rads), engrafted with bone marrow cells (4 × 106) from WT and Δαβ mice, and maintained on sulfamethoxazole- and trimethoprim-supplemented drinking water for 8 weeks prior to analysis. All animal experiments were conducted under IACUC-approved protocols.Δα , CD4 (GK1.5 and RM4–5), CD8 (53–6.7), CD25 (PC61.5), Foxp3 (anti-mouse/rat staining kit), IFN-γ (XMG1.2), IL-4 (BVD6–24G2), IL-17A . Antibodies against the following proteins were used in cell stimulation, Fc receptor blocking, and cytokine neutralization: CD3 (145–2C11), CD28 (37.51), IFN-γ (XMG1.2), IL-12 (C17.8), IL-4 (11B11), CD16/CD32 ; and hamster IgG . Antibodies against the following proteins were used in immunoblotting: ERK (9102), phosphorylated (p-) p38 (9211), p-JNK (9251), p-ERK (9101), p-S6K1 (9205), p-S6 (4858), p-MK2 (3007), p-TSC2 ; p38α ; p38β ; p38γ and p38δ (University of Dundee); JNK ; Foxp3 ; and actin .+ T cells and naïve CD4+ T cells were isolated from the thymus, lymph nodes, and spleen of 6–8-week-old mice using the Pan T cell Isolation Kit and the CD4+CD62L+ T Cell Isolation Kit, respectively (Miltenyi Biotec). Single-cell suspensions obtained from mice were incubated with Fc receptor-blocking anti-CD16/CD32, stained with fluorescent-conjugated antibodies, and analyzed by flow cytometry using FACSCanto and LSRFortessa (BD Biosciences) and FlowJo software (Tree Star).CD3+ T cells from the spleen were stimulated with plate-bound anti-CD3 (10 μg/ml) and soluble anti-CD28 (2 μg/ml). Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled T cells were stimulated with anti-CD3 and anti-CD28 for 3 days, and analyzed by flow cytometry. To analyze Treg suppressive function, lymph node and splenic CD4+CD25+ cells were isolated using the Regulatory T Cell Isolation Kit (Miltenyi Biotec). CFSE-labeled naïve CD4+ T cells were mixed with CD4+CD25+ cells, stimulated with anti-CD3 and anti-CD28 for 3 days, and analyzed for proliferation.Naïve CD4+ T cells from the spleen were incubated with the following agents: Th1, IL-2 (20 ng/ml), IL-12 (10 ng/ml), IFN-γ (10 ng/ml), anti-IL-4 (10 μg/ml); Th2, IL-2 (20 ng/ml), IL-4 (10 ng/ml), anti-IFN-γ (10 μg/ml), and anti-IL-12 (10 μg/ml); and Th17, IL-6 (20 ng/ml), TGF-β (2.5 ng/ml), anti-IFN-γ (2 μg/ml), anti-IL-4 (2 μg/ml), soluble anti-CD28 (1 μg/ml), soluble anti-CD3 (0.25 μg/ml), and plate-bound anti-hamster IgG (0.12 μg/ml). After 5 days of differentiation, cells were treated with phorbol myristate acetate (50 ng/ml) and ionomycin (1 μm) in the presence of brefeldin A (10 μg/ml) for 5 h and analyzed by flow cytometry. For Treg cell induction, naïve CD4+ T cells were stimulated with anti-CD3 and anti-CD28 in the presence of IL-2 (20 ng/ml), TGF-β (5 ng/ml), anti-IFN-γ (10 μg/ml), and anti-IL-4 (10 μg/ml) for 5 days. Cells were treated with SC409 (10 μm), PF3644022 (10 μm), and rapamycin (0.1 μm) where indicated.To induce T cell differentiation into Th effector cells, naïve CD4Whole cell lysates were prepared and analyzed by immunoblotting as described . Immunobp values were obtained with the unpaired, two-tailed Student's t test unless otherwise indicated.Data values are expressed as mean ± S.E. or mean ± S.D. in vitro experiments. M. H., P. R., K. O., K. G., J. S. C. A., and J. M. P. interpreted data. All authors reviewed the results and approved the final version of the manuscript.M. H., K. O., K. G., J. S. C. A., and J. M. P. designed the study and wrote the report. M. H., H. H., T. P., P. R., R. V. S., G. Y. J-A., Y. S., M-K. C., J. S., R. K. C. V., and J. M. P. generated and analyzed p38 and MK KO mice. M. H., T. P., P. R., R. V. S., R. K. C. V., and J. M. P. performed


Crystal structure, packing, and FTIR characterization of 5,5′-dihydroxymethyl-3,3′-biisoxazole and 4,4′,5,5′-tetrahydroxymethyl-3,3′-biisoxazole are reported. 8H8N2O4 (1), is composed of two trans planar isoxazole rings [r.m.s deviation = 0.006 (1) Å], each connected with a methyl hydroxyl group. Similarly, the structure of 4,4′,5,5′-tetrakis(hydroxymethyl)-3,3′-biisoxazole, C10H12N2O6 (2), is composed of two planar isoxazole rings [r.m.s. deviation = 0.002 (1) Å], but with four hydroxymethyl groups as substituents. Both molecules sit on a center of inversion, thus Z′ = 0.5. The crystal structures are stabilized by networks of O—H⋯N [for (1)] and O—H⋯O hydrogen-bonding interactions [for (2)], giving rise to corrugated supramolecular planes. The isoxazole rings are packed in a slip-stacked fashion, with centroid-to-centroid distances of 4.0652 (1) Å for (1)  and of 4.5379 (Å) for (2) .The molecular structure of 5,5′-bis(hydroxymethyl)-3,3′-biisoxazole, C These compounds have been synthesized recently in our laboratory as useful precursors to a new class of energetic materials. The addition of nitric acid to the title compounds results in nitrate esterification, yielding the energetic materials biisoxazolebis(methylene dinitrate) (3) and biisoxazoletetrakis(methyl nitrate) (4), where a nitrate functional group replaces the hydrogen atom in the hydroxyl groups  exhibit a trans planar configuration [r.m.s deviation = 0.0009 (1) Å], suggesting a delocalized aromatic π system. The C4 atom is nearly coplanar with the ring (atom-to-mean plane distance = 0.006 Å), whereas the C4—O2 bond is twisted slightly out of the plane, as evidenced by the torsion angles C2—C1—C4—O2 = −13.3 (2)° and O1—C1—C4—O2 = 167.55 (11)°. Atoms C1/C4/O2 form a plane that subtends a dihedral angle of 12.72 (1)° with respect to the isoxazole ring. Similarly, the isoxazole rings of (2) are nearly planar [r.m.s deviation = 0.002 (1) Å]; however, the corresponding O2—C4 bond is twisted more out of plane than that of compound (1), as evidenced by the magnitude of the torsion angle O2—C4—C1—O1 = −54.93 (11)°. For comparison, the torsion angle formed by atoms O3—C5—C2—C1 is −110.02 (11)°. The atoms O2/C4/C1 and O3/C5/C2 form planes subtending dihedral angles of 53.78 (8) and 69.37 (7)° with respect to the isoxazole ring. Superimposition of the ring atoms of both structures  exhibits a weak intramolecular interaction involving atoms O3—H3A and N1iii The title compounds exhibit molecular structures typical of biisoxazole derivatives. Fig. 11) and (2), respectively, and Tables 11) displays hydrogen bonding between the oxygen atoms O2, belonging to the hydroxy groups, and the N1 atoms of the isoxazole rings of adjacent molecules, generating a supramolecular framework parallel to (i = 2.8461 (15) Å; symmetry code: (i) x − y + z − 2) forms a network of hydrogen bonds involving the hydroxy groups O2—H2A and O3—H3A of adjacent molecules, so that each OH group acts both as donor and acceptor [see Table 2i = 2.694 (1) Å; symmetry code: (i) −x + 1, y + z + ii = 2.790 (1) Å; symmetry code: (ii) x + 1, −y + z + Intermolecular hydrogen bonding plays a key role in the stabilization of the crystal structures of the title compounds. Figs. 31) reveals a slip-stacked geometry of the rings in the b-axis direction, with centroid-to-centroid distances of 4.0652 (1) Å and plane-to-plane shifts of 2.256 (2) Å. In contrast, in compound (2) the rings are stacked along the a-axis direction, with centroid-to-centroid distances of 4.5379 (4) Å and plane-to-plane shifts of 2.683 (2) Å.The crystal structure of    and (2), respectively, with the hydrogen atoms in the OH groups replaced by NO2 moieties. A superimposition of the respective isoxazole rings of compound (1) and (3) yields an r.m.s. deviation of 0.004 Å  the O1 and O2 atoms are in a trans conformation with respect to the C1—C4 bond, whereas in (3) the corresponding O atoms are in a cis conformation. In (1), the plane encompassing the atoms O2, C4, and C1 forms a dihedral angle of 12.72 (1)° with respect to the mean plane of the isoxazole ring, in contrast to a value of 66.8 (2)° in (3) for the corresponding atoms. A similar comparison between (2) and (4) yields an r.m.s. deviation of 0.01 Å for the superimposition of the isoxazole rings, and dihedral angles of 53.78 (8) and 69.37 (7)° for (2)  compared to those of 84.54 (14) and 84.81 (18)° or 79.19 (15) and 82.32 (17)° for (4)  and (4) is that the former exhibit hydrogen bonding, which contributes to the stability of their crystal structure.A search of the Cambridge Structural Database  Fig. 5B. The moet al., 2017a1), a saturated solution of sodium bicarbonate was added to a solution of dichloroglyoxime (30 g), propargyl alcohol (55.2 ml), and methanol (1900 ml) over 6 h. Once the reaction was complete, the product was stirred for an additional 10 h and the remaining solvent evaporated. A yield of 75% was obtained after the product was washed with distilled water, collected by Büchner filtration, and then dried. Compound (2) was prepared by adding dropwise a dichloroglyoxime and butyl alcohol solution (0.8 M) to a refluxing solution comprising NaHCO3 (6.7 g), 2-butyne-1,4-diol (13.72 g), and butyl alcohol (200 ml). Once the reaction was complete, the product was cooled to room temperature and the remaining solvent evaporated. Then, the product was washed with distilled water, filtered, and dried, resulting in a yield of 68%. Slow solvent evaporation of the title compounds in methanol yielded suitable single crystals for the X-ray diffraction experiments at 150K. We note the title compounds have nearly the same density (1.596 vs 1.597 Mg m−3), given that their molecular mass and cell constants are quite different.The synthesis of the title compounds has been reported recently (Wingard 1) and (2) recorded with a Nicolet iS50 spectrophotometer, using attenuated total reflectance. The intense peak frequencies (cm−1) are listed as follows: Compound (1): 3371.83, 3126.65, 1596.96, 1415.14, 1360.62, 1268.13, 1237.16, 1080.70, 1058.61, 1026.40, 993.24, 929.53, 901.95, 828.87, 746.83, 653.69, 621.96, and 424.11. Compound (2): 3234.89, 1623.59, 1456.55, 1418.41, 1354.66, 1261.30, 1185.44, 1128.41, 1046.52, 1011.82, 984.07, 964.14, 931.24, 906.80, 764.50, 725.86, 641.00, 576.90, 475.85, and 449.97.Fig. 6(1) were refined using a riding model with C—H = 0.93 or 0.98 Å and Uiso(H) = 1.2Ueq(C) and O—H = 0.74–0.85 Å and Uiso(H) = 1.5Ueq(O), whereas for compound (2) all the hydrogen atoms were refined independently including isotropic displacement parameters.Crystal data, data collection, structure solution and refinement details are summarized in Table 310.1107/S2056989018000828/xi2007sup1.cifCrystal structure: contains datablock(s) 1, 2. DOI: 10.1107/S2056989018000828/xi20071sup3.hklStructure factors: contains datablock(s) 1, 2. DOI: 10.1107/S2056989018000828/xi20072sup2.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989018000828/xi20071sup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018000828/xi20072sup5.cmlSupporting information file. DOI: 1816712, 1816711CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II ions has an O4N2 coordination environment.Two cadmium coordination polymers have two-dimensional polymeric structures in which monomeric units are joined by bridging acetate and benzenediamine ligands. Each of the Cd 2-acetato-κ8O:O′-bisdicadmium], [Cd2(CH3COO)4(C6H8N2)2]n, (I), and poly[[(μ2-acetato-κ2O:O′)cadmium] hemihydrate], {[Cd(CH3COO)2(C6H8N2)]·0.5H2O}n, (II), have two-dimensional polymeric structures in which monomeric units are joined by bridging acetate and benzenediamine ligands. Each of the CdII ions has an O4N2 coordination environment. The coordination geometries of the symmetry-independent CdII ions are distorted octahedral and distorted trigonal antiprismatic in (I) and distorted antiprismatic in (II). Both compounds exhibit an intralayer hydrogen-bonding network. In addition, the water of hydration in (II) is involved in interlayer hydrogen bonding.Poly[tetra-μ II is able to substitute for ZnII in the active sites of zinc-containing enzymes and to interfere with the metabolism of CaII, which are the primary reasons for its toxicity 2O,O′ and μ2-acetato-κ2O:O′ modes are found in (II)Metal–organic frameworks (MOFs) have received much attention because of their many potential applications including gas storage, catalysis, chemical sensors and molecular separation , 45.92 (17) and 45.92 (17)° [average 42 (6)°] using opposite triangular faces O2O4iN2iv and N2iiO4ivO2vii 2N:N′ benzene-1,3-diamine nitrogen atom (O1O2N2i) forming one of the trigonal faces and two κ2O:O′ acetate ligand oxygen atoms and a nitrogen atom from a κ2N:N′ benzene-1,3-diamine (O3O4iiN1) forming the other trigonal face et al., 2014et al., 2004et al., 2007et al., 2006et al., 2008abet al., 2013et al., 2006et al., 2007et al., 2012et al., 2004et al., 2005et al., 2000et al., 2013et al., 2012Examples of cadmium coordination polymers with carboxylate ligands and that exhibit two-dimensional sheet structures have been reported (Li −1): 3278 (w), 1532 (s), 1504 (s), 1405 (s). 1H NMR : 1.87 , 6.35 , 6.35 .213 mg (0.924 mmole) cadmium acetate hydrate were dissolved in 10 mL of ethanol. With stirring, 204 mg (1.89 mmol) of benzene-1,2-diamine were added and the resulting solution was refluxed for 2 h. A white precipitate formed, which was isolated by filtration and dried under vacuum. The yield was quantitative (310 mg). Selected IR bands : 3425 (b), 3329 (s) 3328 (b), 3137 (m), 1520 (s), 1505 (s), 1400 (s). 1H NMR : 1.83 , 5.78 , 6.64 . 13C NMR : 22.1, 100.5, 103.6, 129.6, 149.5, 178.0.230 mg (1.00 mmol) cadmium acetate hydrate were dissolved in 10 mL of ethanol. 217 mg (2.01 mmol) benzene-1,3-diamine were added with stirring. The solution was gently refluxed for 2 h. After chilling the reaction mixture in an ice bath, the precipitate was filtered and dried under vacuum. A yield of 248 mg (71%) was obtained. Selected IR bands  = 1.5Ueq(C). All other C—H hydrogen atom isotropic displacement parameters were set using the approximation Uiso(H) = 1.2Ueq(C). The N—H bond lengths were restrained to 0.88 Å in (I)Uiso(H) was refined freely for the amine and water hydrogen atoms, except that for (II)Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989016017382/pj2037sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989016017382/pj2037Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016017382/pj2037Isup4.molSupporting information file. DOI: 10.1107/S2056989016017382/pj2037IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989016017382/pj2037IIsup5.molSupporting information file. DOI: 1512964, 1512963CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The asymmetric unit contains two crystallographically independent cages of 2,5-diazabicyclo[2.2.1]heptane, each cage being protonated at the two nitrogen sites. The overall charge balance is maintained by four crystallogrphically independent bromide ions. In the crystal, the components of the structure are linked  S,4S)-2,5-diazoniabicyclo[2.2.1]heptane dibromide, C5H12N22+·2Br−. The asymmetric unit contains two crystallographically independent cages of 2,5-diazabicyclo[2.2.1]heptane. Each cage is protonated at the two nitrogen sites. The overall charge balance is maintained by four crystallographically independent bromide ions. In the crystal, the components of the structure are linked via a complex three-dimensional network of N—H⋯Br hydrogen bonds.The cage of 2,5-diazabicyclo[2.2.1]heptane is frequently employed in synthetic chemistry as a rigid bicyclic counterpart of the piperazine ring. The 2,5-diazabicyclo[2.2.1]heptane scaffold is incorporated into a variety of compounds having pharmacological and catalytic applications. The unsubstituted parent ring of the system, 2,5-diazabicyclo[2.2.1]heptane itself, has not been structurally characterized. We herein report on the molecular structure of the parent ring in (1 Moreover, the parent ring of unsubstituted 2,5-diazabicyclo[2.2.1]heptane has not been structurally characterized. In the framework of current research on caged heterocyclic systems -2,5-diazoniabicyclo[2.2.1]heptane dibromide (1).Derivatives of the bicyclic nucleus of 2,5-diazabicyclo[2.2.1]heptane comprise a wide family of biochemically active compounds  Å], whereas the distances between the bridgehead C atoms C1⋯C4 [2.220 (4) Å] and C1A⋯C4A [2.226 (4) Å] are statistically the same (see the Supporting information). Therefore, in spite of bridge-imparted rigidity, the hexagonal ring of 2,5-diazabicyclo[2.2.1]heptane can be affected by some geometric distortions. The framework of 2,5-diazabicyclo[2.2.1]heptane is frequently considered to be the bicyclic counterpart of piperazine where the occurrence of the C1–C7–C4 bridge imparts rigidity to the hexagonal ring , there are just 14 structures which comprise this bicyclic system in the Cambridge Structural Database -2,5-diazabicyclo[2.2.1]heptanes and provided the first structure determination of the 1,2,5-substituted derivative. Lauteslager et al. -Diazabicyclo[2.2.1]heptane dihydrobromide (1) was obtained from Sigma–Aldrich and found to be analytically pure . NMR spectra  are consistent with the previously published data : δ = 4.67 , 3.65–3.57 , 2.29 . 13C{1H} NMR : δ = 56.36 , 47.09 , 34.73 . Crystals of 1 suitable for structural study were obtained by slow evaporation of a saturated aqueous solution at room temperature.(1i.e. those involved in hydrogen bonding) were freely refined whereas hydrogen atoms at all carbon centers were treated with fixed Uiso(H) = 1.2Ueq(C) and riding coordinates (C—H = 0.97–0.98 Å).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017015870/lh5858sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017015870/lh5858Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017015870/lh5858Isup3.molSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017015870/lh5858Isup4.cmlSupporting information file. DOI: 1578911CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The decomposition of chloroform in the presence of oxygen and moisture provides HCl, which leads to the breakdown of nanojars to the title trinuclear copper(II) pyrazolate complex, and possibly CuII ions and free 4-iodopyrazole. CuII ions, in turn, act as catalyst for the accelerated decomposition of chloroform, ultimately leading to the complete breakdown of nanojars. The crystal structure presented here provides the first structural description of a trinuclear copper(II) pyrazolate complex with iodine-substituted pyrazoles. In contrast to related trinuclear complexes based on differently substituted 4-R-pyrazoles , the [Cu3(μ-4-I-pz)3Cl3] core in the title complex is nearly planar. This difference is likely a result of the presence of the iodine substituent, which provides a unique, novel feature in copper pyrazolate chemistry. Thus, the iodine atoms form halogen bonds with the terminal chlorido ligands of the surrounding complexes [mean length of I⋯Cl contacts = 3.48 (1) Å], leading to an extended two-dimensional, halogen-bonded network along (-110). The cavities within this framework are filled by centrosymmetric 1,4-dioxane solvent molecules, which create further bridges via C—H⋯Cl hydrogen bonds with terminal chlorido ligands of the trinuclear complex not involved in halogen bonding.Crystals of bis(tetrabutylammonium) di-μ This is due to the acidity of the conjugate acid, the triethylammonium cation (pKa = 10.75 in H2O), which would form in the process 2[{Cu(μ-OH)(μ-4-I-pz)}nCO3] (n = 27–31) 2[Cu3(μ3-Cl)2(μ-4-I-pz)3Cl3]·0.5dioxane  . What is surprising though is the large amount of chloride formed in a relatively short period of time (ca 48 chloride ions per nanojar). Chloroform preserved with ethanol (0.5–1%), such as the one used here for crystal growing, is much more stable than the pure form and it does not decompose at a significant rate. This points to a decomposition catalyzed by the dissolved nanojars, possibly aided by light. A search of the literature shows that various classes of compounds have been found to catalyze the photodecomposition of chloroform (μ-4-Ipz)nCO3]2– + 5nHCl → n[Cu3(μ3-Cl)2(μ-4-Ipz)3Cl3]2– + (2n − 6) H3O+ + (n + 9) H2O + 3CO2 (n = 27–31).3. The two capping Cl atoms impart an overall 2– charge to the complex, which is balanced by two tetrabutylammonium counter-ions. Other bond lengths and angles within the Cu3(3μ-Cl)2(μ-4-I-pz)3Cl3 complex are similar to the ones found in related complexes ; I⋯Cl distances and C–I⋯Cl angles are shown in Table 1A/Cl1B and I1′ and I3′. The formation of the extended halogen-bonded network might account for the near-planarity of the title complex, as opposed to related complexes with unsubstituted or differently substituted 4-R-pyrazoles °], creating further bridges within the two-dimensional framework.The intermolecular distances between iodine substituents of the pyrazole units and the terminal chlorine atoms of adjacent complexes are less than the sum of the van der Waals radii 2[{Cu(μ-OH)(μ-4-I-pz)}nCO3] (n = 27–31) was described earlier 2[{Cu(μ-OH)(μ-4-I-pz)}nCO3] (20 mg).The synthesis of (Bu2CH3 groups of one tetrabutylammonium counter-ion and another CH2CH3 group of the other counter-ion are disordered over two positions (60/40); C—H bond-length restraints were used for the disordered C atoms. Residual electron density of 3.52 eÅ−3 is found at 0.83 Å from heavy atom I3, due to Fourier truncation ripples.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901601536X/gk2666sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698901601536X/gk2666Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901601536X/gk2666Isup3.cdxSupporting information file. DOI: 1507382CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


LINE-1 (L1) retrotransposons are a notable endogenous source of mutagenesis in mammals. Notably, cancer cells can support unusual L1 retrotransposition and L1-associated sequence rearrangement mechanisms following DNA damage. Recent reports suggest that L1 is mobile in epithelial tumours and neural cells but, paradoxically, not in brain cancers.Alu-Alu recombination event adjacent to an L1. These mutations included PCR validated intronic events in MeCP2 and EGFR. Despite sequencing L1 integration sites at up to 250× depth by RC-seq, we found no tumour-specific, endonuclease-dependent L1 insertions. Whole genome sequencing analysis of the tumours carrying the MeCP2 and EGFR L1 mutations also revealed no endonuclease-dependent L1 insertions. In a complementary in vitro assay, wild-type and endonuclease mutant L1 reporter constructs each mobilised very inefficiently in four cultured GBM cell lines.Here, using retrotransposon capture sequencing (RC-seq), we surveyed L1 mutations in 14 tumours classified as glioblastoma multiforme (GBM) or as a lower grade glioma. In four GBM tumours, we characterised one probable endonuclease-independent L1 insertion, two L1-associated rearrangements and one likely These experiments altogether highlight the consistent absence of canonical L1 retrotransposition in GBM tumours and cultured cell lines, as well as atypical L1-associated sequence rearrangements following DNA damage in vivo.The online version of this article (doi:10.1186/s13100-016-0076-6) contains supplementary material, which is available to authorized users. Ninety-ary GBM) . Primaryary GBM) . To dateary GBM) –6. Defecary GBM) , 8. DespAlu retrotransposons, in trans [Alu elements can also participate in DNA rearrangements driven by recombination [L1 retrotransposons are endogenous mutagens known to cause sporadic disease, including cancer . A full-in trans –27. L1 abination , 29. Altbination –38 and nbination –42, sevebination –32, 35.We hypothesised that L1-associated DNA rearrangements in GBM might occur via recombination or an atypical retrotransposition mechanism and therefore may lack the TPRT hallmarks required for L1 insertion recognition by previous genomic analyses. Alternatively, we considered that L1 insertions in GBM could be restricted to sub-clonal and highly heterogeneous events. We therefore applied deep retrotransposon capture sequencing (RC-seq) , 42 to 1Tissues were obtained from 14 patients undergoing surgical removal of a brain tumour at the Department of Clinical Neurosciences, Western General Hospital, Edinburgh, UK. All patients gave informed consent for tumour and peri-tumoural tissue removed in the normal course of surgery, and blood obtained intra-operatively, to be used for research. Ethical approval for the study was granted to P.M.B. by the East of Scotland Research Ethics Service (SR018). Tissue designation as ‘tumour’ or ‘adjacent brain’ was determined at the time of sampling based on pre-operative imaging, intra-operative image guidance and macroscopic inspection. Blood was sampled from 9 patients  tumour and adjacent brain from patients #1-#7, ii) tumour and adjacent brain from patients #8-#14 and iii) blood from patients #6-#12 and #14 were hybridised using a third generation (V3) RC-seq capture protocol involving only two optimised, custom locked nucleic acid (LNA) oligonucleotide probes  respectively targeting the 5′ and 3′ ends of L1.4  Fig. 1a1a. LNA phttps://github.com/adamewing/tebreak/tree/f7f01c1) with settings –mincluster 2, −minclip 30, −minq 1. Briefly, TEBreak aligned RC-seq reads against the human reference genome (hg19) using BWA-MEM [http://broadinstitute.github.io/picard/). Non-duplicate reads that aligned partially to the reference genome but had ≥30 nt soft-clipped from either end were retained; clipped ends were then aligned against L1.4 using the same BWA-MEM settings. Split-read mappings joining an L1 to the reference genome were then clustered and annotated for the presence of TSDs or deletions. Clusters with at least two reads supporting a consistent breakpoint were output in VCF format and further post-processed using the summary.py script included in the TEBreak distribution. This further filtered candidates by ensuring a consistent breakpoint between BWA-MEM and BLAT [RC-seq read data were analysed using TEBreak  following manufacturer’s instructions for total RNA isolation. Quantification was performed using NanoDrop 1000 . 2 μg total RNA was treated with DNase I  and used as template for cDNA synthesis with SuperScript III Reverse Transcriptase  following manufacturer’s instructions. 2 μg total RNA was processed as described with no reverse transcriptase added to the cDNA synthesis reaction for further use as negative control (RT-). cDNA from adjacent brain tissue was diluted to final concentrations of 1:5, 1:10, 1:20, 1:40 and 1:80 and used to generate a standard curve for each primer set. Samples were diluted to 1:20 final concentration for qRT-PCR. Real time PCRs were performed using SensiFast SYBR Lo-ROX kit  and run on a ViiA 7 Real-Time PCR System  with standard curve experiment analysis settings. Negative control qRT-PCRs were performed using water as template  and 2 μl of RT- reaction; no amplification was detected. MeCP2 isoforms (NM_004992) were assessed using 5′-GAGGCGAGGAGGAGAGAC and 5′-TGGTAGCTGGGATGTTAGGG as forward primers for isoforms 1 and 2, respectively, and 5′-GCAGAGTGGTGGGCTGAT as a common reverse primer to amplify 154 nt of isoform 1 and 161 nt of isoform 2. Additionally, 143 nt of the MeCP2 exon 4, present in both isoforms, were amplified using 5′-CAGAGGAGGCTCACTGGAGA as forward primer and 5′-GGCATGGAGGATGAAACAAT as reverse primer. EGFR (NM_005228) was amplified on the 5′UTR (amplicon of 156 nt) and the junction between exons 11 and 12 (176 nt) with the following primers:5′-ACAGCTTTGAAGAGAGCAGTGGTT, 5′-AGTCTGCCCGTTCTCAGATCTL1 ORF2: 5′-TGCGGAGAAATAGGAACACTTTT, 5′-TGAGGAATCGCCACACTGACTL1 (L1.4) expression was assessed by targeting 61 nt of the 5′UTR and 85 nt of the ORF2 with the primers: L1 5′UTR:TBP, 5′-GCAAGGGTTTCTGGTTTGCC, 5′-GGGTCAGTCCAGTGCCATAAACTB, 5′-AGAAAATCTGGCACCACACC, 5′-TAGCACAGCCTGGATAGCAA156nt of TATA-binding protein mRNA  and 173 nt of beta actin mRNA  were amplified with the following primers:2) are as follows: MeCP2 isoform 1: −3.312, 29.329, 0.95; MeCP2 isoform 2: −2.946, 29.578, 0.943; MeCP2 exon 4: −3.796, 27.991, 0.897; EGFR 5′UTR: −4.057, 30.028, 0.931; EGFR exon 11–12 junction: −4.660, 28.317, 0.985; TBP: −3.754, 27.208, 0.973; L1 5′UTR: −3.094, 21.209, 0.996; L1 ORF2: −3.11, 18.836, 0.9992; ACTB: −3.954, 22.606, 0.999.Standard curve parameters for qRT-PCR  or ACTB (for L1). Statistical analysis was performed with Prism5 (GraphPad Software), applying a cDNA from patient #2 tumour was used as template for RT-PCR to amplify possible L1-MeCP2 chimeric transcripts. The following chimeric variants were tested; Exon 1–L1, using a forward primer within exon 1 5′-GAGGCGAGGAGGAGAGAC and a reverse primer within the new L1, 5′-CACCAGCATGGCACATGTAT. Exon 2–L1, forward primer (5′-TGGTAGCTGGGATGTTAGGG) in exon 2 and reverse primer within L1 (5′-CACCAGCATGGCACATGTAT). L1-Exon 3, with the L1 primer as a reverse primer (5′-GCACATTGTGCAGGTTAGTTAC) and a forward primer within exon 3, (5′-GCAGAGTGGTGGGCTGAT).5 ng of genomic DNA from patient #2 adjacent brain and tumour were used as a template for a PCR to amplify the deleted region within MeCp2 using 5′-AAATTAGCCAGGCGTGGTG as forward primer within the deleted region and 5′-TCCTGTTTTGTCTTACGTCTTGA as reverse primer downstream of the deleted region. The PCR conditions were as follow; 1U MyTaq DNA polymerase , 1× MyTaq Reaction Buffer, 2 μM primers and 5 ng template DNA in a 25 μL reaction volume with the following cycling conditions: 3 min at 92 °C, then 35 cycles of 15 s at 92 °C, 15 s at 56 °C and 15 s at 72 °C, followed by a single extension step at 72 °C for 10 min. PCR amplicons were resolved in a 2 % agarose gel and scanned . Amplicons present on the scanned picture was quantified using Image Studio Lite version 4 software (LI-COR Biosciences). Values were corrected for background and normalised to adjacent brain value.200 ng of genomic DNA extracted from tumour and adjacent brain tissues was bisulfite converted using EZ DNA Methylation-Lightning Kit  following manufacturer’s instructions. After purification, 2 μL was used as template for a PCR reaction using L1-Bis-F and L1-Bis-R primers as described by Shukla et al. . PCR reaIllumina libraries (TruSeq Nano DNA sample preparation kit) were generated from patient #2 tumour and adjacent brain and, for patient #8, tumour and blood genomic DNA. Libraries had an insert size of ~300 nt and were sequenced on an Illumina HiSeq X Ten platform . Reads were aligned to hg19 using BWA-MEM (parameters –Y –M –R < read group name>) and sorted using SAMtools . PCR dupGBM cell lines were purchased from American Type Culture Collection  and grown in a humidified, 5 % CO2 incubator at 37 °C in the complete media as described by the provider. DBTRG-05MG  cells were grown in ATCC-formulated RMPI-1640 medium  supplemented with 10 % foetal bovine calf serum , non-essential amino acids  and 100 U/mL penicillin, 0.1 mg/mL streptomycin  to generate complete media. M059J  cells were grown in media containing a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s F12 Medium  supplemented with 10 % foetal bovine calf serum, non-essential amino acids and 100 U/mL penicillin, 0.1 mg/mL streptomycin. LN18  and LN229  were grown in Dulbecco’s Modified Eagle’s Medium  supplemented with 5 % foetal bovine calf serum and 100 U/mL penicillin, 0.1 mg/mL streptomycin. HeLa cells were grown in a humidified, 5 % CO2 incubator at 37 °C in the complete media, Dulbecco’s Modified Eagle’s Medium  supplemented with 10 % foetal bovine calf serum and 100 U/mL penicillin, 0.1 mg/mL streptomycin.NotI-Hf and BstZ17I restriction enzymes  at 37 °C for 2 h to obtain L1.3-D205A and the JJ-NotI-Hf/BstZ17I backbone. This backbone was also treated with alkaline phosphatase  for 30 min at 37 °C to use for subsequent cloning. Purified L1.3-D205A was ligated to the JJ-NotI-Hf/BstZ17I backbone using T4 ligase  for 2 h at room temperature generating JJ-L1.3-D205A. To obtain wild-type L1.3 (L1.3 WT) [EcoRI , NotI and BstZ17I simultaneously (2 h at 37 °C). Fragments corresponding to L1.3-NotI/EcoRI, L1.3-D205A-NotI/EcoRI, L1.3-EcoRI/BstZ17I and L1.3-D702A-EcoRI/BstZ17I were gel purified. L1.3-NotI/EcoRI and L1.3-EcoRI/BstZ17I were ligated to JJ-NotI-Hf/BstZ17I backbone to obtain JJ-L1.3 plasmid. JJ-L1.3-D205A-D702A was generated by ligation of L1.3-D205A-NotI/EcoRI and L1.3-D702A-EcoRI/BstZ17I to the JJ-NotI-Hf/BstZ17I backbone. Ligations were transformed in One Shot TOP10 Chemically Competent E. coli bacteria , and plated in LB-agar  and 100 μg/ml ampicillin . All plasmid sequences were confirmed by capillary sequencing.Plasmids carrying i) an L1 EN mutant (pCEP4-L1.3D205A)  and ii) L1.3 WT)  and L1.32O to 0.5 μg/μL. GBM cells were seeded in 6-well dishes in their respective complete media to ~25 % confluence. Cells were transfected at the time of seeding with FuGENE HD transfection reagent  following the manufacturer’s protocol using 1:4 DNA: FuGENE ratio. Each transfection well received 1 μg plasmid DNA, 4 μL FuGENE reagent and 2 mL of complete media. Media was changed 24 h post transfection and selection with blasticidin S HCl  began 4 days post transfection. Cells were selected for antibiotic resistance for 10 days using a final concentration of 5 μg/ml for HeLa, DBTRG-05MG and LN229 and 2 μg/mL for LN18 and M059J.13-14 days post transfection, cells were washed twice with 1xPBS and fixed and stained as described by Moran et al. [Plasmid DNA was purified on maxiprep columns  and diluted in sterile dHTransfection efficiency for each plasmid was calculated by flow cytometry . GBM cell lines were co-transfected as described above with 0.5 μg of each JJ construct and 0.5 μg of pCAG-GFP (plasmid that constitutively expresses GFP). 72 h post-transfection, cells were harvested and re-suspended in 1× PBS. Propidium iodide  was added to the samples for identification of dead cells. GFP positive cells were gated based on fluorescence of untransfected cells and transfection efficiency calculated as the percentage of GFP positive cells using FlowJo 10.0.8 software   gene by RC-seq that was confirmed by PCR  reductions in tumoural expression of both of the two main MeCP2 transcript isoforms  L1 mRNA abundance in patient #2 tumour versus adjacent brain   Fig. . Replicap < 0.0001, t-test)  Fig. , a majorst) Fig. . As EGFRst) Fig. . Given tTo place the PCR validated L1-associated mutations in MeCP2 and EGFR into a broader context of genomic abnormality, we analysed WGS data from patients #2 and #8 (Additional file As noted above, the tumour sample from patient #8 showed a remarkable amplification of EGFR (>50 fold, Fig. To assess whether GBM cell lines support canonical or ENi L1 retrotransposition in vitro we performed an established cultured cell retrotransposition assay  on HeLa These experiments reveal rare L1-associated mutations caused by recombination or L1 ENi retrotransposition in GBM tumours, accompanied by an absence of TPRT-driven L1 insertions. Endonuclease-independent L1 insertions have been reported by several prior studies employing engineered L1 reporter constructs in cultured cancer cells or cells otherwise deficient for DNA damage repair factors , 72, 73,Alu:Alu recombination. Notably, the Alu-flanked L1PA2 donor sequence, located on chromosome 9, was 5′ truncated and did not encode a viable L1 ORF2p. The involvement of an older L1 family in a tumour-specific DNA rearrangement event is reminiscent of one of the earliest L1 mutations detected in cancer, in that case affecting the myc locus in breast tumour [Alu during ENi L1 mobilisation in trans, in agreement with a previous observation where 71 % of detected ENi L1 mobilisation events involved non-L1 DNA [Although identified at first by RC-seq as a potential L1 mobilisation event, further characterisation of the MeCP2 L1 mutation indicated a probable DNA rearrangement event mediated by t tumour . A lessen-L1 DNA , and invn-L1 DNA , 73, 75.Notably, the EGFR L1 mutation provides an interesting example because, despite not having a direct effect on gene activity, it is one of very few tumour-specific L1 mutations noted in a major oncogene or tumour suppressor since the first such example was discovered more than 20 years ago by Miki et al. .de novo L1 insertions in this context is intriguing, given frequent somatic L1 mobilisation via TPRT in neural cells [Previous studies employing high-throughput sequencing reported no tumour-specific L1 insertions in brain tumours –32, 35. al cells –42, inclal cells . This coAlthough we conclusively find that L1 mobilisation is a rare event in GBM, our discovery of atypical L1-associated mutations in MeCP2 and EGFR demonstrates that L1 can otherwise contribute to GBM genome abnormality in key loci regulating neural cell differentiation and proliferation. Future experiments are required to ascertain whether this phenomena correlates with patient prognosis and whether potential DNA damage caused by chemotherapy or radiotherapy  activate


Cotreatment with mevalonate (MEV), farnesol (FOH), geranylgeraniol (GGOH), or water-soluble cholesterol (Chol-PEG) was employed to determine whether the statin-dependent myotoxicity resulted from the lower cholesterol levels or the attenuated synthesis of intermediates of mevalonate pathway. Our findings demonstrated that while GGOH fully reverted the statin-mediated cell viability in proliferating myoblasts, Chol-PEG exclusively rescued MβCD-induced toxicity in myocytes. Statins caused loss of prenylated RAP1, whereas the GGOH-dependent positive effect was accompanied by loss of nonprenylated RAP1. Geranylgeranyltransferases are essential for muscle cell survival as inhibition with GGTI-286 could not be reversed by GGOH cotreatment. The increase in cell viability correlated with elevated AKT 1(S463) and GSK-3β(S9) phosphorylations. Slight increase in the levels of autophagy markers  was found in response to GGOH cotreatment. Autophagy rose time-dependently during myogenesis and was inhibited by statins and MβCD. Statins and MβCD also suppressed myogenesis and neither nonsterol isoprenoids nor Chol-PEG could reverse this effect. These results point to GGOH as the principal target of statin-dependent myotoxicity, whereas plasma membrane cholesterol deposit is ultimately essential to restore viability of MβCD-treated myocytes. Overall, this study unveils for the first time a link found between the GGOH- and Chol-PEG-dependent reversal of statin- or MβCD-mediated myotoxicity and cytoprotective autophagy, respectively.The present study investigated the cytotoxic effects of statins (atorvastatin (ATR) and simvastatin (SIM), resp.) and methyl-beta-cyclodextrin (M Atherosclerosis and cardiovascular diseases are main causes of mortality in humans . Statinsβ, downstream substrate inactivated by AKT1, activates TSC1/TSC2. Altogether, activation of PI3-K/AKT signaling should indirectly (by inhibiting TSC1/TSC2 and GSK-3β) stimulate RAP1A activity.The RAS subfamily of small molecular weight GTPases—active when bound to GTP and inactive in the GDP-bound state , 10—is kThere is evidence that statin-induced muscle toxicity is associated with inhibition of protein geranylgeranylation . PrenylaSkeletal muscle growth and regeneration (myogenesis) take place in two subsequent phases. After an initial stage of cell proliferation, myoblasts withdraw from the cell cycle and differentiate and fuse to form multinucleated myotubes and muscle fibers , 19. TheTaken together, the aim of our study was to bridge the gap of understanding molecular mechanism of statin-dependent myopathy.trans-geranylgeraniol (GGOH), farnesol (FOH), ubiquinol (UBOH), methyl-beta-cyclodextrin (MβCD), poly(ethylene-glycol 600)-cholesterol conjugate (Chol-PEG), poly(ethylene-glycol 600) (PEG), 3--2-5-diphenyltetrazolium bromide (MTT), bovine serum albumin (BSA), and acridine orange were purchased from Sigma Aldrich, Saint Louis, MO, USA. C-100-Dolichol  was obtained from Larodan, Karolinska Institutet Science Park, Solna, Sweden, and bisbenzimide fluorochrome from Molecular Probes, Eugene, OR, USA.Atorvastatin, a cell-permeable, highly potent, and competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, was purchased from Calbiochem, Merck-Millipore . Simvastatin, (R)-mevalonic acid lithium salt, all-v/v) heat-inactivated fetal bovine serum , penicillin/streptomycin , gentamicin sulfate , and Fungizone/amphotericin B , and grown until 70–80% confluence. Spontaneous differentiation by growth factor withdrawal was induced replacing growth medium with differentiation medium (DM) consisting of DMEM supplemented with 2% (v/v) heat-inactivated horse serum  and the same antibiotic/antimycotic cocktail as the GM and further incubation for up to 5 days. Muscle cells and syncytia were harvested on days 1, 3, and 5 of myogenic differentiation time points at which they displayed different phenotypes. At day 1, cells exhibited proliferating myoblasts, at day 3, differentiating myotubes, and at day 5, differentiated myotubes.The murine skeletal muscle cell line C2C12  was obta50, Supplementary μM, day 3—46 μM, and day 5—36 μM; SIM: day 1—125 μM, day 3—10 μM, and day 5—7.5 μM. MβCD was added at 2.7 mM, 1.9 mM, and 1.1 mM final concentrations at day 1, day 3, and day 5, respectively.Treatment of the cultures with the different experimental factors tested in the current work was performed 24, 72, or 120 hours prior to cell harvesting based on the respective half-maximal inhibitory concentrations . The discrepancy is explained by the time of experiment which lasted merely 5 days (in vivo myogenesis lasts considerably longer—at least few weeks). As reported by us, the ICμL per well) and color evaluated at 570 nm with TECAN 200 multiplate reader .Cell viability was assessed evaluating the ability of cells to convert soluble MTT -2-5-diphenyltetrazolium bromide) into an insoluble purple formazan as described . Briefly2/95% air incubator) with AO (1 μg/mL) and HO33342 (10 μg/mL), both dissolved in DMEM without phenol red, and analyzed in an Olympus IX-71 inverted fluorescence microscope. Abundance of AVO was calculated as red to green fluorescence intensity ratio (R/G-FIR) in each microscopic field, as previously described  amounted to F = 67.52, p < 0.0001 for ATR; F = 9.014, p = 0.0006 for SIM; F = 3.349, p = 0.0521 for MβCD. Treatment: ATR, ATR + MEV, ATR + GGOH, ATR + FOH, and ATR + Chol-PEG  = 0.2542, p = 0.9520); SIM, SIM + MEV, SIM + GGOH, SIM + FOH, and SIM + Chol-PEG  = 0.2754, p = 0.9423); MβCD, MβCD + MEV, MβCD + GGOH, MβCD + FOH, and MβCD + Chol-PEG  = 0.5688, p = 0.7228). Interaction: F = 8.344, p < 0.0001 for ATR; F = 9.014, p = 0.0006 for SIM; F = 1.055, p = 0.42 for MβCD. Densitometric analysis of proteins on Western blots. Two-way ANOVA test for P-GSK-3β-S9 versus GSK-3β optical density ratio followed by Bonferroni's multiple comparisons was employed to analyze the data. The results of [time ] amounted to F = 6.199, p = 0.0059 for ATR; F = 65.48, p < 0.0001 for SIM; and F = 22.25, p < 0.0001 for MβCD. Treatment: ATR, ATR + MEV, ATR + GGOH, ATR + FOH, and ATR + Chol-PEG  = 41.29, p < 0.0001); SIM, SIM + MEV, SIM + GGOH, SIM + FOH, and SIM + Chol-PEG  = 0.6259, p = 0.7074); MβCD, MβCD + MEV, MβCD + GGOH, MβCD + FOH, and MβCD + Chol-PEG  = 0.1876, p = 0.9568). Interaction: F = 3.455, p = 0.0033 for ATR; F = 2.057, p = 0.0568 for SIM; F = 0.8854, p = 0.5705 for MβCD. GGOH protected proliferating myoblasts and differentiating myotubes but not differentiated myotubes from the cytotoxic effects of statins and MβCD which are evidenced by elevated P-AKT1-S473 and P-GSK-3β-S9  . ATR or SIM was administered in the listed concentration at each day of differentiation: ATR: proliferating myoblasts—76 μM, differentiating myotubes—46 μM, and differentiated myotubes—36 μM; SIM: proliferating myoblasts—87 μM, proliferating myoblasts—6 μM, and proliferating myoblasts—3 μM or vehicle control (0.1% DMSO or 2% HS DMEM) without or with selected mevalonate pathway intermediate . (a) Atorvastatin  inhibited the Beclin 1 protein expression levels in differentiated myotubes irrespective to cotreatment except the Chol-PEG (1 mM) cotreatment which reversed statin-dependent effect during in vitro myogenesis. On the contrary, Chol-PEG further lessens Beclin 1 protein level reduced by now in ATR-treated differentiating myotubes. In turn, FOH (10 μM) was the sole factor which diminished Beclin 1 in proliferating myoblasts. ATR (IC50) significantly reduced the amount of MAP LC3-I/IIb compared to untreated cells in differentiating myotubes. MEV overturned ATR effect, whereas GGOH markedly elevated the MAP LC3-IIb expression level even above the untreated control. Neither ATR nor isoprenoids could affect MAP LC3-I/IIb protein expression levels in proliferating myoblasts and differentiated myotubes. SIM does not seem to be as effective as ATR in MAP LC3-I/IIb processing. (b) For comparison, SIM-dependent effect on Beclin 1 was apparently more accentuated in differentiating myotubes. In contrast to ATR, however, GGOH protected SIM-dependent decline of MAP LC3-IIb in differentiated myotubes  and GGOH (10 μM) cotreatment increased Beclin 1 levels in differentiating myotubes whereas Chol-PEG (1 mM) in differentiated myotubes. MβCD as cholesterol chelator increased MAP LC3-IIb in proliferating myoblasts, and none of the cotreatment affected this effect. Chol-PEG significantly elevated MAP LC3-I/IIb expression levels in differentiated myotubes. Representative blots are shown in (a), (b), and (c) from three independent repeats. Densitometric analysis of proteins on Western blots. Two-way ANOVA test for Beclin 1 versus actin optical density ratio followed by Bonferroni's multiple comparisons was employed to analyze the data. The results of [time ] amounted to F = 34.73, p < 0.0001 for ATR; F = 30.22, p < 0.0001 for SIM; and F = 2.012, p = 0.1557 for MβCD. Treatment: ATR, ATR + MEV, ATR + GGOH, ATR + FOH, and ATR + Chol-PEG  = 0.3566, p = 0.8943); SIM, SIM + MEV, SIM + GGOH, SIM + FOH, and SIM + Chol-PEG  = 30.22, p = 0.0822); MβCD, MβCD + MEV, MβCD + GGOH, MβCD + FOH, and MβCD + Chol-PEG  = 0.2727, p = 0.0229). Interaction F = 1.139, p = 0.3707 for ATR; F = 2.168, p = 0.0845 for SIM; F = 0.2727, p = 0.9194 for MβCD. Densitometric analysis of proteins on Western blots. Two-way ANOVA test for MA PLC-II3b to MAP LC3-Ib optical density ratio followed by Bonferroni's multiple comparisons was employed to analyze the data. The results of [time ] amounted to F = 1.135, p = 0.3358 for ATR; F = 19.56, p < 0.0001 for SIM; and F = 12.30, p = 0.0002 for MβCD. Treatment: ATR, ATR + MEV, ATR + GGOH, ATR + FOH, and ATR + Chol-PEG  = 1.782, p = 0.1748); SIM, SIM + MEV, SIM + GGOH, SIM + FOH, and SIM + Chol-PEG  = 4.149, p = 0.0422); MβCD, MβCD + MEV, MβCD + GGOH, MβCD + FOH, and MβCD + Chol-PEG  = 3.995, p = 0.0229). Interaction F = 1.139, p = 0.3707 for ATR; F = 2.168, p = 0.0845 for SIM; F = 0.6263, p = 0.777 for MβCD. Densitometric analysis of proteins on Western blots. Two-way ANOVA test for RAP1A (NP) versus actin optical density ratio followed by Bonferroni's multiple comparisons was employed to analyze the data. The results of [time ] amounted to F = 69.12, p < 0.0001 for ATR; F = 17.89, p < 0.0001 for SIM; and F = 8.616, p = 0.0048 for MβCD. Treatment: ATR, ATR + MEV, ATR + GGOH, ATR + FOH, and ATR + Chol-PEG  = 32.63, p < 0.0001); SIM, SIM + MEV, SIM + GGOH, SIM + FOH, and SIM + Chol-PEG  = 28.30, p < 0.0001); MβCD, MβCD + MEV, MβCD + GGOH, MβCD + FOH, and MβCD + Chol-PEG  = 0.3290, p = 0.8785). Interaction: F = 5.224, p = 0.0002 for ATR; F = 2.708, p = 0.0146 for SIM; F = 1.178, p = 0.3885 for MβCD. Densitometric analysis of proteins on Western blots. Two-way ANOVA test for RAP1B (P + NP) versus actin optical density ratio followed by Bonferroni's multiple comparisons was employed to analyze the data. The results of [time ] amounted to F = 11.00, p = 0.0003 for ATR; F = 11.43, p = 0.0002 for SIM; and F = 1.077, p = 0.3567 for MβCD. Treatment: ATR, ATR + MEV, ATR + GGOH, ATR + FOH, and ATR + Chol-PEG  = 0.9608, p = 0.4852); SIM, SIM + MEV, SIM + GGOH, SIM + FOH, and SIM + Chol-PEG  = 1.042, p < 0.4397); MβCD, MβCD + MEV, MβCD + GGOH, MβCD + FOH, and MβCD + Chol-PEG  = 0.1311, p = 0.9822). Interaction: F = 1.604, p = 0.1472 for ATR; F = 1.397, p = 0.2251 for SIM; F = 0.3767, p = 0.9449 for MβCD. Densitometric analysis of proteins on Western blots. Two-way ANOVA test for RAP1A/RAP1B (P + NP) versus actin optical density ratio followed by Bonferroni's multiple comparisons was employed to analyze the data. The results of [time ] amounted to F = 715.96, p < 0.0001 for ATR; F = 23.47, p < 0.0001 for SIM; and F = 15.75, p = 0.0004 for MβCD. Treatment: ATR, ATR + MEV, ATR + GGOH, ATR + FOH, and ATR + Chol-PEG  = 29.38, p < 0.0001); SIM, SIM + MEV, SIM + GGOH, SIM + FOH, and SIM + Chol-PEG  = 1.41, p = 0.3286); MβCD, MβCD + MEV, MβCD + GGOH, MβCD + FOH, and MβCD + Chol-PEG  = 0.09, p = 0.9918). Interaction: F = 41.07, p < 0.0001 for ATR; F = 2.08, p = 0.0968 for SIM; F = 0.76, p = 0.6644 for MβCD. ATR treatment increased nonprenylated RAP1A GTPase protein expression levels throughout the myogenesis  and farnesyl pyrophosphate (FPP) . In the myotubes . (c) MβCogenesis . This efogenesis . Chol-PEogenesis . Althougogenesis .50 concentrations of ATR inhibited expression levels of Beclin 1 protein in differentiated myotubes irrespective to the cotreatment agent with the exception of Chol-PEG which completely reversed the statin-dependent effect  with AO to assess autophagy revealed that ATR either alone or together with MEV, GGOH, FOH, or Chol-PEG did not affect AVO formation (Supplementary βCD . MβCD si50) at days 1, 3, and 5 of myogenesis. While separate treatment with GGOH did not affect cell viability, addition of GGTI-286, alone or in combination with GGOH, significantly changed MTT reduction levels highlighting a fundamental role for geranylgeranyltransferases but not GGOH itself (p < 0.05). Overall, these data point to protein prenylation as a possible mechanism for GGOH protection against statin-induced impairment of myoblast viability during myogenesis and suggest that GGOH becomes harmful when geranylgeranyltransferases are withdrawn from the prenylation process.An inhibitor of the protein geranylgeranyltransferases, GGTI-286, was administered at half-maximal cell viability inhibitory concentration (ICH itself . The addβCD (IC50) . ATR or SIM was administered in the listed concentration at each day of differentiation: ATR: day 1—76 μM, day 3—46 μM, and day 5—36 μM; SIM: day 1—87 μM, day 3—6 μM, and day 5—3 μM. MβCD was added to give the final concentration: day 1—2.7 mM, day 3—1.9 mM, and day 5—1.1 mM or vehicle control (0.1% DMSO or 2% HS DMEM) without or with selected mevalonate pathway intermediate . No myogenin expression was detected at day 1 regardless of treatment. (a) Atorvastatin  inhibited myogenin expression level at day 3 but not at day 5. Only GGOH (10 μM) partially reversed the ATR effect by accelerating myogenin amount at day 3. In turn, at day 5, GGOH cotreatment caused moderate fall in myogenin expression. (b) Correspondingly, simvastatin  decreased myogenin expression levels at day 3 without any noticeable effect at day 5. Similarly to ATR, addition of GGOH (10 μM) reduced myogenin expression level at day 5. (c) Methyl-beta-cyclodextrin  markedly reduced myogenin amount at day 3, even though it could hardly affect the expression levels at day 5. A representative blots are shown in (a), (b), and (c) from three independent repeats. Densitometric analysis of proteins on Western blots. Two-way ANOVA test for myogenin versus actin optical density ratio followed by Bonferroni's multiple comparisons was employed to analyze the data. The results of [time ] amounted to F = 6.444, p = 0.0050 for ATR; F = 86.45, p < 0.0001 for SIM; and F = 127.7, p < 0.0001 for MβCD. Treatment: ATR, ATR + MEV, ATR + GGOH, ATR + FOH, and ATR + Chol-PEG  = 0.8277, p = 0.5675); SIM, SIM + MEV, SIM + GGOH, SIM + FOH, and SIM + Chol-PEG  = 1.385, p = 0.2870); MβCD, MβCD + MEV, MβCD + GGOH, MβCD + FOH, and MβCD + Chol-PEG  = 3.212, p = 0.0453. Interaction: F = 0.1894, p = 0.9972 for ATR; F = 4.333, p = 0.0007 for SIM; F = 4.305, p = 0.0016 for MβCD. ATR, SIM, and MβCD at their respective (IC50) concentrations significantly reduced myogenin levels in day 3 and 5 differentiating myotubes  or MβCD (IC50) . ATR or SIM was administered in the listed concentration at each day of differentiation: ATR: day 1—76 μM, day 3—46 μM, and day 5—36 μM; SIM: day 1—87 μM, day 3—6 μM, and day 5—3 μM. MβCD was added to give the final concentration: day 1—2.7 mM, day 3—1.9 mM, and day 5—1.1 mM or vehicle control (0.1% DMSO or 2% HS DMEM) without or with selected mevalonate pathway intermediate . (a) Atorvastatin  did not affect MyHC protein expression levels during in vitro myogenesis. Eventually, mevalonate , farnesol , and Chol-PEG (1 mM) cotreatment significantly elevated MyHC protein at day 1. (b) Simvastatin  did not change MyHC protein levels irrespective to time and type of treatment. (c) Also, methyl-beta-cyclodextrin  could not effect MyHC, but in contrast to ATR and SIM, cotreatment with GGOH (10 mM) augmented MyHC expression levels at days 3 and 5. Representative blots are shown in (a), (b), and (c) from three independent repeats. Densitometric analysis of proteins on Western blots. Two-way ANOVA test for MyHC versus actin optical density ratio followed by Bonferroni's multiple comparisons was employed to analyze the data. The results of [time ] amounted to F = 108.6, p < 0.0001 for ATR; F = 1.707, p = 0.1997 for SIM; and F = 155.9, p < 0.0001 for MβCD. Treatment: ATR, ATR + MEV, ATR + GGOH, ATR + FOH, and ATR + Chol-PEG  = 12.76, p < 0.0001); SIM, SIM + MEV, SIM + GGOH, SIM + FOH, and SIM + Chol-PEG  = 2.149, p < 0.1118); MβCD, MβCD + MEV, MβCD + GGOH, MβCD + FOH, and MβCD + Chol-PEG  = 6.297, p = 0.0222). Interaction F = 10.51, p < 0.0001 for ATR; F = 1.163, p = 0.3546 for SIM; F = 8.718, p = 0.0004 for MβCD.Myosin heavy chain protein is a representative product of the myogenin-mediated activation of the ogenesis . Total p50 concentrations) and MβCD hardly affected myotube index (MI) at day 1 of differentiation  in skeletal muscle cells undergoing differentiation process divided into three basic steps: postmitotic proliferation, cell fusion, and myotube enlargement. To this aim, different experimental factors were added (or not) starting at day 1 and continued to day 3 or to day 5 of cell differentiation and the subsequent evaluations were referred to as changes in “proliferating myoblasts,” “differentiating myotubes,” and “differentiated myotubes,” respectively . Upon assessment of the half-maximal cytotoxic concentration (IC50), the action of mevalonate pathway intermediates and water-soluble form of cholesterol in cotreatment experiments could clearly be demonstrated. Initially, the nonsterol isoprenoids were selected according to their reliance on mevalonate input blocked by statins , geranylgeraniol (GGOH), farnesol (FOH), dolichol (DOH), and ubiquinol (UBOH)). Finally, the molecular studies focused on the role of MEV, GGOH, and FOH, compounds that were confronted with water-soluble form of cholesterol, Chol-PEG.In the present study, we examined the differential effect of statins  and the cholesterol chelator  βCD-challenged myoblasts. Thus, GGOH was able, once more, to reverse—at least in part—the statin-dependent AKT1 dephosphorylation. Changes in GSK-3β phosphorylation and protein expression levels followed the pattern observed for AKT1 at every step of myogenesis (the lower the level of T-AKT1 and P-AKT1 (S473), the lower the level of T-GSK-3β and P-GSK-3β (S9)). The mechanisms by which statins inhibit AKT1 kinase activity remain to be completely elucidated although they seem not to result from a direct action of statins and most likely involve blunted insulin and/or insulin-like growth factor (IGF) signaling due to impaired prenylation and/or N-glycosylation of upstream signaling molecules for AKT1 [Moving further to characterized the cell signaling mechanisms involved in these interactions, we studied the AKT1 kinase to GSK-3l muscle . Providil muscle  but alsol muscle , 39. Ourfor AKT1 . Insulinfor AKT1 . Since RβCD to proliferating myoblasts .The present studies combined the visualization of the autophagy process through vital staining of myocytes with the assessment of red fluorescence-emitting acidic vacuolar organelles (AVO) and green fluorescent non-AVO. The calculated R/GFIR index  revealedlication . The choβCD effects on RAP1 prenylation, the protein expression levels were accomplished in their nonprenylated versus prenylated form. As shown in Figure  were noted  limits differentiation by promoting myoblast proliferation, but once cell divisions are completed, the accelerated myogenin expression also induced by prenylated RAP1 promotes cell fusion. This mechanism is consistent with the numerous known biological functions of RAP1 including adhesion, spreading, and migration, all fundamental for muscle syncytium formation . Moreoveeatments . ExplanaβCD + GGOH cotreatments in differentiating myotubes  and tested ATR and SIM in their ability to reduce HMG-CoA reductase activity in comparison with MβCD, a compound that binds and removes cholesterol from plasma membrane. In order to assess which mevalonate pathway intermediate or end product is capable to reverse statin and MβCD toxicity, we tested mevalonate (MEV), geranylgeraniol (GGOH), farnesol (FOH), and water-soluble cholesterol (Chol-PEG). Geranylgeraniol, a cell-permeable analogue of geranylgeranyl pyrophosphate (GGPP), reversed statin-induced myotoxicity in proliferating myoblasts and differentiating myotubes but not in MβCD-treated myocytes. GGOH-mediated positive effects were entirely dependent on Geranylgeranyltransferases (GGTs) activities. In turn, Chol-PEG restored viability to control levels only in MβCD cotreated myocytes. GGOH administration increased expression levels of prenylated RAP1A/1B GTPase. This protein in prenylated form also elevated Beclin1 and MAP LC3-IIb in muscle cells affected by ATR or SIM. Chol-PEG did the same in MβCD-treated differentiated myotubes. GGOH had a broader protective effect against statin-induced myotoxicity than MEV, FOH, or Chol-PEG. Most of the GGOH- or Chol-PEG-mediated stimulations of myogenesis were seemingly associated with cytoprotective autophagy. AKT1 and GSK-3β kinase phosphorylations increased in parallel to viability promoted by GGOH.The aim of this study was to elucidate the molecular mechanisms of statin-induced myotoxicity as successful strategy to prevent statin-derived side effects is lacking. Statins competitively inhibit HMG-CoA reductase leading to significant decreases in the intracellular synthesis of mevalonate, geranylgeraniol, farnesol, ubiquinone, dolichol, and cholesterol. The current work established the respective half-maximal inhibitory concentrations  phosphorylation.Lower myotoxicity is reflected by the respective increase in AKT 1 (S463) and GSK-3Geranylgeranyltransferases (GGTs) control myocyte viability through GGOH, which in excess is likely myotoxic.βCD-impaired autophagy.Cytoprotective autophagy is elevated in myocytes during myogenesis. MEV, GGOH, and Chol-PEG are able to reverse statin- or MβCD-challenged myocytes.Plasma membrane cholesterol is fundamental for the survival of MβCD, and nonsterol isoprenoids or Chol-PEG could not reverse this effect.Muscle differentiation is impaired by statins or M


In the crystal, hydrogen-bonded layers are linked by π–π and C—H⋯F⋯π interactions.The  17H17F6N2O+·C8H7O3−·0.5H2O, comprises a pair of pseudo-enantiomeric  piperidin-1-ium cations, two carboxylate anions and a water molecule of crystallization. The cations have similar conformations approximating to a letter, L: one of them shows disorder of its –CF3 group over two sets of sites in a 0.775 (3):0.225 (3) ratio. Distinctive conformations are found for the anions, one with the carboxylate group lying to one side of the plane through the phenyl ring and the other where the oxygen atoms lie to either side of the plane. In the latter, an intramolecular hydroxy-O—H⋯O(carboxylate) charge-assisted hydrogen bond is found. The packing features extensive O—H⋯O,N hydrogen bonding, often charge-assisted; C—H⋯π interactions are also formed. The hydrogen bonding results in the formation of five distinctive supramolecular synthons and assembles molecules in the ac plane. The quinolinyl rings lie to either side of the layer and inter-digitate with layers on either side, are approximately parallel to the b axis and are connected by π–π [inter-centroid separation = 3.6904 (18) Å] as well as C—F⋯π(quinolinyl) interactions to consolidate the three-dimensional crystal. The dominance of the conventional hydrogen bonding in the molecular packing is confirmed by an analysis of the Hirshfeld surface.The asymmetric unit of the title salt, C R*,S*)-quinolin-4-yl](hydroxymethyl)piperidin-1-ium chloride salt; see Scheme for the chemical diagram of the cation, also known as mefloqinium. This salt, racemic erythro-mefloquine hydrochloride, has been used as an anti-malarial drug since 1971  have been described where the supramolecular association led to chains in each case, but these were sustained by distinct hydrogen-bonded synthons  with the N1-cation having an S-configuration at the C12 atom and an R-configuration at C13 and therefore being assigned as the [(−)-erythro-mefloquinium] cation. The N3-cation, with chirality at the C29 and C30 atoms being R and S, respectively, is assigned as [(+)-erythro-mefloquinium]. As anticipated, protonation during crystallization leads to a piperidin-1-ium cation, as confirmed by the pattern of hydrogen bonding, which is discussed below in Supramolecular features. Each cation comprises an essentially planar quinolinyl residue attached to a piperidinium residue (with a chair conformation) via a methine link. The dihedral angle between the quinolinyl-NC5 ring plane and the best plane through the piperidinium ring is 71.91 (16)° indicating an almost perpendicular relationship so that the cation adopts an l-shape; the equivalent dihedral angle for the N3-cation is 80.58 (17)°. This assignment is also supported by the values of the C2—C3—C12—C13 and C19—C20—C29—C30 torsion angles of −100.4 (3) and 108.1 (3)°, respectively. The hydroxyl-O and piperidinium-N atoms lie to the same side of the piperidinium ring, being gauche across the methine-C—C(methine) bond with N2⋯O1 = 3.019 (4) Å and O1—C12—C13—N2 = 73.3 (3)° for the N1-cation; the equivalent values for the N3-cation are 2.931 (4) Å and −70.7 (3)°, respectively. The similarity in the two cations is emphasized in the overlay diagram shown in Fig. 2The asymmetric unit of (I)S-configuration at the C36 atom and an R-configuration at atom C44 of the second independent anion. The confirmation of deprotonation is found in the near equivalence of the C35—O3, O4 [1.260 (4) and 1.263 (4) Å] and of the C43—O6, O7 [1.223 (5) and 1.246 (5) Å] bond lengths. As evidenced from the overlay diagram shown in Fig. 4syn to a carboxylate-O atom as seen in the O3—C35—C36—O5 and O7—C43—C44—O8 torsion angles of 151.9 (3) and 17.3 (6)°, respectively. However, it is only in the O6-anion that the aforementioned hydrogen bond is formed to close a five-membered {⋯HOC2O} loop, Table 1The anions in (I)2 H atoms of the N1-cation forms charge-assisted hydrogen bonds to the water molecule of crystallization and to the O3-carboxylate atom, whereas those of the N3-cation interact with the hydroxy-O8 and carboxylate-O4 atoms.In addition to considerable conventional hydrogen bonding, often charge-assisted, there are other intermolecular interactions at play in the molecular packing  interaction in the O6-anion. The hydroxy group of the O3-anion forms a hydroxy-O—H⋯O(carboxylate) link between the anions. Both carboxylate-O3, O4 atoms accept hydrogen bonds from piperidinium-N—H H atoms whereas the carboxylate-O5, O6 atoms form interactions with piperidinium-N—H and anion–hydroxyl-H H atoms, respectively. The carboxylate-O3 and O7 atoms each form two hydrogen bonds with the additional interactions involving water-H atoms. Finally, as just mentioned, the water molecule forms two donor interactions with carboxylate-O atoms, accepts a hydrogen bond from a piperidinium-N—H H atom and also accepts a contact from a quinolinyl-C—H atom.a, the largest synthon is sustained exclusively by O—H⋯O hydrogen bonding, being a centrosymmetric 22-membered {⋯OCO⋯HOH⋯OC2OH}2 ring. Four other rings are formed mediated by hydrogen bonding but the only remaining centrosymmetric synthon features two bridging piperidinium-N—H H atoms, which link water- and carboxylate-O atoms to generate a 12-membered {⋯HNH⋯OH⋯O}2 synthon. The three remaining synthons do not have symmetry. The smallest, nine-membered {⋯HNC2OH⋯OCO} abuts the 12-membered synthon just described and shares a common N—H bond. The nine-membered synthon is connected on the other side by an 12-membered ring featuring the second piperidinium-N—H2 group, i.e. {⋯HNC2OH⋯OH⋯OC2O}. Portions of both of the nine- and 12-membered synthons participate in the formation of a larger 15-membered synthon which involves both piperidinium-N—H2 groups, i.e. {⋯HNH⋯OC2O⋯HNH⋯OH⋯O⋯HO}; one of the O—H⋯O links is the intramolecular hydroxy-O—H⋯O(carboxylate) hydrogen bond. A tight methylene-C—H⋯π(anion-phenyl) interaction is also noted, Table 1ac plane with the quinolinyl residues lying to either side in the b-axis direction and in orientations enabling inter-digitation. Interactions between rings are of the type π–π, occurring between quinolinyl-bound (C21–C26) and i rings with an inter-centroid separation of 3.6904 (18) Å and angle of inclination of 8.70 (15)°; symmetry code (i): 1 − x, 1 − y, 1 − z. A variety of C—F⋯π(quinolinyl) interactions provide additional links in the inter-layer region. A view of the unit-cell contents is shown in Fig. 5b.The just described hydrogen bonding gives rise to a number of cyclic synthons. Referring to Fig. 5Crystal Explorer , respectively; the faint-red spots near the piperidinium-N4, Fig. 6c, and carboxylate-O4 atoms, Fig. 6e, are due to the presence of comparatively weak N—H⋯O hydrogen bonds. The existence of water-O—H⋯O hydrogen bonds can be viewed as bright-red spots near the H2W and carboxylate-O3 atoms while the other is indicated with dashed lines in Fig. 6e. Finally, the bright-red spots at hydroxyl-H5O, Fig. 6f, and carboxylate-O6, Fig. 6e, provides a link between the anions through O—H⋯O interactions.The Hirshfeld surfaces mapped over 3 groups of the cations indicate their participation in various intermolecular interactions. The faint-red spots near the F1, F7 and F11 atoms shown in Figs. 6a, 6c and 6d, indicate short interatomic F⋯F contacts, Table 2b and Table 2a, 6b and 6d, and from the short interatomic C⋯F contacts listed in Table 2c, 6d and 6e. The faint-red spots present near the methylene-C14—H, Fig. 6b, and anion-phenyl-C42 atoms, Fig. 6e, and short interatomic C⋯H/H⋯C contacts between methylene-H14A and anion atoms C37, C41 and C42, as summarized in Table 2c and 6f, and quantified in Table 2e and 6f, and N1-cation hydrogens H6 and H15A, Figs. 6a and 6b, are also viewed as faint-red spots near these atoms. The immediate environments about the N1- and N3-cations and the anions and water molecule within the dnorm-mapped Hirshfeld surface mediated by the above interactions are illustrated in Fig. 9The faint-red spots near the fluorine atoms of the CFdi and de in the form of two-dimensional fingerprint plots  distribution of points reflected as a saw-tooth with the tips at de + di ∼2.1 Å correspond to a short interatomic piperidinium-H1O⋯H2O contact between hydroxyl-hydrogens of the two independent cations, Table 2de, di) region less than the van der Waals separations, i.e. 2 × 1.2 Å. The 19.2% contributions from O⋯H/H⋯O contacts to the overall surface results from intermolecular O—H⋯O, N—H⋯O and C—H⋯O interactions as well as short interatomic O⋯H/H⋯H contacts in the crystal, Table 2c, a pair of long spikes having tips at de + di ∼1.7 Å and the appearance of green points aligned as a pair of streaks are due to the presence of dominant O—H⋯O and N—H⋯O hydrogen bonds.The combination of d, show a fin-like distribution of points with the edges at de + di ∼2.6 Å resulting from the presence of C—H⋯π interactions and short interatomic C⋯H/H⋯C contacts, as summarized in Table 2de + di ∼2.7 Å and 2.8 Å in a tube-shaped distribution of points in the fingerprint plot delineated into F⋯H/H⋯F contacts, Fig. 10e, arise from short intermolecular F⋯H/H⋯F contacts, Table 2f, the distribution of points in a pencil-tip shape with the tip at de + di ∼2.8 Å represent the short interatomic F⋯F contacts listed in Table 2de + di ∼3.0 Å, in the fingerprint plot delineated into C⋯F/F⋯C contacts, Fig. 10g, and make a 4.6% contribution to the surface. A small 2.3% contribution from C⋯C contacts to the Hirshfeld surface with the parabolic distribution of points, Fig. 10h, around the  distances slightly shorter than their van der Waals radii, i.e. 2 × 1.7 Å, indicate π–π stacking interactions between quinolinyl rings. The presence of π–π stacking interactions between the symmetry-related rings is also indicated through the appearance of red and blue triangle pairs on the Hirshfeld surface mapped with shape-index property identified with arrows in the images of Fig. 11The fingerprint plot corresponding to C⋯H/H⋯C contacts, Fig. 10et al., 2016et al., 2016i.e. contain enantiomeric species that are not related by crystallographic symmetry, meaning they crystallize in one of the 65 Sohncke space groups, which lack inversion centres, rotatory inversion axes, glide planes and mirror planes. This phenomenon is rare for organic species, occurring in just 0.1% of their structures  crystals isolated from the 1:1 reaction of mefloquinium chloride and p-fluorobenzenesulfonyl chloride in the presence of NaOH -erythro-mefloquinium] cations as well as a chloride and p-fluorobenzenesulfonate anions to provide the charge balance. The second example was isolated from the attempted chiral resolution of mefloquine with the carboxylic acid, 3,3,3-trifluoro-2-methoxy-2-phenylpropanoic acid, i.e. (+)-PhC(CF3)(OMe)CO2H. Crystallography showed the triclinic (P1) crystals to comprise the [(+)-erythro-mefloquinium] and [(−)-erythro-mefloquinium] cations with two independent (+)-3,3,3-trifluoro-2-methoxy-2-phenylpropanate anions. Hence, different anions appear to have promoted kryptoracemic behaviour in the chloride/p-fluorobenzenesulfonate salt  in MeOH (10 ml) were mixed at room temperature. The reaction mixture was set aside at room temperature for three days and the resulting colourless slabs collected; M.pt: 434–346 K. IR (KBr disc): 3400–2100 (v br), 1586, 1313, 1190, 1130, 739 cm−1. 13C NMR : δ 21.74, 22.01, 22.38, 44.54, 59.25, 68.39, 73.45, 115.43, 121.22 [J(C—F) = 273.6 Hz], 123.69 [J(C—F) = 272.2 Hz], 126.34, 126.38, 126.52, 127.19 [J(C—F) = 291.3 Hz], 127.53, 128.11. 129.05, 129.76 [J(C—F) = 4.7 Hz], 142.74, 143.03, 146.64 [J(C—F) = 34. 0 Hz], 151.82, 175.75 p.p.m.Solutions of mefloquine (1 mmol) in MeOH (15 ml) and (Uiso(H) = 1.2Ueq(C). The O- and N-bound H atoms were located from difference maps but, refined with O—H = 0.84±0.01 Å and N—H = 0.88±0.01 Å, and with Uiso(H) = 1.2Ueq(N) and 1.5Ueq(O). One reflection, i.e. (3 group was modelled as being disordered over two orientations with a site occupancy ratio 0.775 (3):0.225 (3). The anisotropic displacement parameters for pairs of F atoms were constrained to be equal and restrained to be nearly isotropic. Even so, one atom in particular showed elongated displacement ellipsoids, i.e. the F8 atom, but this was not modelled further. Multiple atomic positions were not discerned for the O6-anion, Fig. 3b. Finally, the maximum and minimum residual electron density peaks of 1.75 and 0.66 eÅ−3, respectively, were located 0.84 Å and 0.35 Å from the H44 and O7 atoms, respectively. Given the strong and directional hydrogen bonding in this region of the molecule, it is likely that the large residual is an artefact of the data.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989016016492/hb7620sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989016016492/hb7620Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016016492/hb7620Isup3.cmlSupporting information file. DOI: 1510084CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


E)-2-{[(2-aminophenyl)imino]methyl}-5-(benzyloxy)phenol (I) and (Z)-3-benzyloxy-6-{[(5-chloro-2-hydroxyphenyl)amino]methylidene}cyclohexa-2,4-dien-1-one (II) are reported. The crystal structures of the molecules are stabilized by N—H⋯O, O—H⋯O and C—H⋯π contacts. DFT calculations on the structure of (II) support the Keto–imine tautomeric form found in the solid state structure. The antioxident properties of both molecules are investigated.The synthesis and structures of ( 20H18N2O2 (I) and C20H16ClNO3 (II), were synthesized from 4-benzyloxy-2-hydroxybenzaldehyde by reaction with 1,2-diaminobenzene for (I), and condensation with 2-amino-4-chlorophenol for (II). Compound (I) adopts the enol–imine tautomeric form with an E configuration about the C=N imine bond. In contrast, the o-hydroxy Schiff base (II), is in the keto–imine tautomeric form with a Z configuration about the CH—NH bond. Neither molecule is planar. In (I), the central benzene ring makes dihedral angles of 46.80 (10) and 78.19 (10)° with the outer phenylamine and phenyl rings, respectively, while for (II), the corresponding angles are 5.11 (9) and 58.42 (11)°, respectively. The molecular structures of both compounds are affected by the formation of intramolecular contacts, an O—H⋯N hydrogen bond for (I) and an N—H⋯O hydrogen bond for (II); each contact generates an S(6) ring motif. In the crystal of (I), strong N—H⋯O hydrogen bonds form zigzag chains of molecules along the b-axis direction. Molecules are further linked by C—H⋯π interactions and offset π–π contacts and these combine to form a three-dimensional network. The density functional theory (DFT) optimized structure of compound (II), at the B3LYP/6–311+G(d) level, confirmed that the keto tautomeric form of the compound, as found in the structure determination, is the lowest energy form. The antioxidant capacities of both compounds were determined by the cupric reducing antioxidant capacity (CUPRAC) process.The title Schiff base compounds, C In compound (II)E with a length of 1.288 (3) Å. In contrast the o-hydroxy Schiff base of (II)Z configuration about the C7=C8 double bond and the molecule adopts the keto–imine tautomeric form, with the N1—C7 bond length being 1.309 (2) Å. Neither molecule is planar: in (I)The molecular structures of compounds (I)A⋯O2i hydrogen bonds, Table 1b-axis direction, Fig. 3Cg2⋯Cg2i hydrogen bonds Table 2A⋯Cg3ii contacts lead to the formation of sheets of molecules parallel to et al. 2016viz. (Z)-6-{2-phenylaminomethylene}-3-hydroxycyclohexa-2,4-dienone -5-(benzyloxy)-2-[(4-nitrophenyl)carbonoimidoyl]phenol reported by us in 2015 -5-benzyloxy-2-{[(4-chlorophenyl)imino]methyl}phenol -5-benzyloxy-2-({[2-(1H-indol-3-yl)ethyl]iminiumyl)methyl)phenolate, which is closely similar to (II)et al., 2010A search of the Cambridge Database . The DFT structure optimization of (II)EHOMO and ELUMO of −5.622 and −2.234 eV, respectively.DFT quantum chemical calculations were performed on molecule (II)II–Nc] (CUPRAC) process  inhibition (IC50) in the CUPRAC assay is small for both compounds in comparison to that for butylated hydroxytoluene (BHT) that was used as a positive control. In Table 4The antioxidant activity profiles of (I)Compound (I)vacuo. The residue was recrystallized from ethanol, yielding yellow block-like crystals on slow evaporation of the solvent. The purity of the compound was determined from its NMR spectrum . The azomethine proton appears in the 8.5–8.6 p.p.m.range, while the imine bond is characterized in the 13C NMR spectrum with the imine C and the C atom bound to the OH group appearing in the 161.58–163.20 p.p.m.range. 1H NMR: δ = 6.6–7.6 , δ = 13.5 , δ = 4 , δ = 5.1 . 13C NMR: 70.22, 127.66, 127.73, 128.32, 128.8, 140.66, 161.58, 163.02, 163.2.1,2-Diaminobenzene (1 equiv.) and 4-benzyloxy-2-hydroxybenzaldehyde (1 equiv.) in ethanol (15–20 ml) were refluxed for 1 h, the solvent was evaporated in Compound (II)vacuo. The residue was recrystallized from ethanol, yielding orange block-like crystals on slow evaporation of the solvent. The purity of the compound was detemined by its NMR spectrum . 1H NMR: δ = 6.5–7.7 , δ = 8.5–8.6 , δ = 5.1 . 13C NMR: 55.6, 128.2, 128.7, 133.3, 136.4, 141.4, 159.69, 162.82, 163.77.2-Amino-4-chloroyphenol (1 equiv.) and 4-benzyloxy-2-hydroxybenzaldehyde (1 equiv.) in ethanol (20 ml) were refluxed for 30–60 min, the solvent was evaporated in Uiso(H) = 1.5Ueq(O). In compound (II)Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989018005662/sj5553sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989018005662/sj5553Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989018005662/sj5553IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989018005662/sj5553Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018005662/sj5553IIsup5.cmlSupporting information file. DOI: 1836250, 1836249CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: