We further demonstrated that CD137 costimulation was essential for Vγ9Vδ2-T cell activation, proliferation, survival and effector functions. In humanized mice reconstituted with human peripheral blood mononuclear cells, CD137 costimulation with a recombinant human CD137L protein boosted the therapeutic effects of pamidronate against influenza virus. Our study provides a novel strategy of targeting CD137 to improve the efficacy of Vγ9Vδ2-T cell-based immunotherapy.Influenza epidemics and pandemics are constant threats to global public health. Although strategies including vaccines and antiviral drugs have achieved great advances in controlling influenza virus infection, the efficacy of these strategies is limited by the highly frequent mutations in the viral genome and the emergence of drug-resistant strains. Our previous study indicated that boosting the immunity of human Vγ9Vδ2-T cells with the phosphoantigen pamidronate could be a therapeutic strategy to treat seasonal and avian influenza virus infections. However, one notable drawback of γδ-T cell-based immunotherapy is the rapid exhaustion of proliferation and effector responses due to repeated treatments with phosphoantigens. Here, we found that the expression of CD137 was inducible in Vγ9Vδ2-T cells following antigenic stimulation. CD137 Vγ9Vδ2-T cells can be specifically activated in an HLA-unrestricted manner by small nonpeptidic phosphoantigens, which are metabolites of isoprenoid biosynthesis pathways.13 Pharmacological compounds, such as the aminobisphosphonates pamidronate (PAM) and zoledronate, which are commonly used for the treatment of osteoporosis and Paget’s disease, can induce the activation and expansion of human Vγ9Vδ2-T cells.15 Previously, we demonstrated that phosphoantigen-activated Vγ9Vδ2-T cells have antiviral activities against human and avian influenza viruses mediated by killing virus-infected monocyte-derived macrophages (MDMs), inhibiting virus replication, and promoting the production of virus-specific antibodies.20 Recently, we further reported that PAM can control influenza diseases by expanding the Vγ9Vδ2-T cell population in humanized mice.22 However, the application of PAM for the treatment of influenza is limited by the rapid exhaustion of the proliferation and effector responses of Vγ9Vδ2-T cells resulting from repeated treatments with the phosphoantigens. Although the underlying mechanisms of this exhaustion are still not clear, the suboptimal stimulation or lack of optimal costimulation may explain it. Therefore, detailed characterizations of the costimulatory factors required for sustained activation and survival of Vγ9Vδ2-T cells are required to solve this problem.23γδ-T cells, as innate-like T lymphocytes, represent a minor but crucial population in the immune system.24 The interaction of CD137 with its major ligand, CD137L, has been shown to result in the survival, expansion, and differentiation of αβ-T cells, particularly CD8+ T cells, into effectors and the establishment of long-term memory.27 Agonistic anti-CD137 monoclonal antibodies (mAbs) have been shown to enhance the efficacy of vaccines against influenza virus and poxvirus by enhancing CD8+ or CD4+ T cell responses.29 However, whether CD137 has similar costimulatory effect on γδ-T cells remains unknown.CD137 , a membrane-bound receptor that belongs to the tumor necrosis factor receptor superfamily, is an inducible T cell costimulatory molecule.In this study, we demonstrated critical roles for CD137-CD137L interactions in the proliferation, survival and effector functions of Vγ9Vδ2-T cells in response to influenza virus infection in vitro and in vivo. Using a recombinant human SA-hCD137L protein containing the extracellular domains of human CD137L (hCD137L) fused to a core streptavidin (SA) molecule, we demonstrated that SA-hCD137L is an efficient adjuvant for PAM therapy for influenza virus infection that functions by improving the antiviral activity of human Vγ9Vδ2-T cells in humanized mice. Our study suggests the novel strategy of targeting CD137 to improve the antiviral activity of human Vγ9Vδ2-T cells.+ cells after 24 h of coculture with influenza virus-infected MDMs  and cell death-related receptor ligands  than their CD137- counterparts. Importantly, the expression of CD107a, a marker associated with the degranulation of NK cells and cytotoxic T lymphocytes (CTLs),30 was significantly higher in CD137+ Vγ9Vδ2-T cells than CD137- Vγ9Vδ2-T cells after coculture with influenza virus-infected MDMs  and antiviral cytokines (IFN-γ) than their CD137− counterparts . CD137+ and CD137− Vγ9Vδ2-T cell subsets were sorted after activation by an anti-γδ-TCR antibody in vitro, and then highly purified (>95%) CD137+, CD137− or whole Vγ9Vδ2-T cells were adoptively transferred into the influenza virus-infected mice on days 2, 4, and 6 post infection  fused to a core streptavidin (SA) molecule was generated. The addition of the recombinant SA-hCD137L protein to a coculture of whole Vγ9Vδ2-T cells with influenza virus-infected MDMs significantly enhanced the expression of CD107a, cytolytic granule molecules (perforin and granzyme B) and an antiviral cytokine (IFN-γ) in the Vγ9Vδ2-T cells  and an antiviral cytokine (IFN-γ) in the Vγ9Vδ2-T cells Fig. . In cont+PI−: early apoptosis; 27.43 ± 1.03%, AV+PI+: late apoptosis) in Vγ9Vδ2-T cells activated by an anti-γδ-TCR antibody for 24 h than did an mIgG1 control   induced by PAM. Compared with control treatment, blocking CD137 resulted in a significant decrease in Vγ9Vδ2-T cells in terms of both the cell frequency Fig.  and absois) Fig. , which i32 were injected intraperitoneally (i.p.) with the recombinant SA-hCD137L protein and PAM 3 days after influenza virus infection ion Fig. . During ion Fig. . Furtherion Fig. . To deteion Fig. . As show+ Vγ9Vδ2-T cell population exhibited an enhanced capability to treat influenza when employed as an adoptive γδ-T cell therapy. Furthermore, we demonstrated that a tetrameric form of a recombinant human CD137L protein, SA-hCD137L, could boost the effect of PAM therapy on influenza by costimulating Vγ9Vδ2-T cells. Our study provides a relatively effective strategy to treat influenza virus by targeting CD137 to improve the antiviral activity of human Vγ9Vδ2-T cells.The ability of CD137 signaling to evoke robust αβ-T cell effector responses has been extensively demonstrated in infectious disease models. However, the role of CD137 signaling in Vγ9Vδ2-T cells remains poorly understood in the context of influenza virus infection. In this study, we demonstrate for the first time that CD137 ligation enhances Vγ9Vδ2-T cell proliferation, survival and antiviral activity against influenza virus. The CD13734 hepatitis C, cytomegalovirus, HIV, lymphocytic choriomeningitis virus, and poxviruses.35 As shown in previous reports on αβ-T cells, CD137 signaling can inhibit activation-induced cell death (AICD), promote cell proliferation and survival, and enhance effector cell function.36 Here, we also found that engagement of CD137 could provide costimulatory signals to Vγ9Vδ2-T cells to enhance proliferation, inhibit apoptosis and increase antiviral activity in vitro and in vivo, suggesting that CD137 can provide costimulatory signals to both αβ- and γδ-T cells.Several studies have shown that CD137 plays a critical role in αβ-T cell-mediated antiviral immune responses against influenza,37 direct comparison of the effector functions of CD137+ and CD137− Vγ9Vδ2-T cell fractions has not been performed. Here, we found that CD137+ Vγ9Vδ2-T cells displayed more potent antiviral activity than their CD137− counterparts in vitro and in vivo. The cytolytic phenotype of CD137+ effector Vγ9Vδ2-T cells suggests that CD137 may be a marker of potent effector Vγ9Vδ2-T cells  and cell death-related receptor ligands  than their CD137− counterparts  complex to induce effective antitumor immune responses.41 SA-mCD137L induces less lymphadenopathy and splenomegaly than antibody therapy, suggesting that SA-mCD137L has a higher therapeutic index. Here, we generated streptavidin-conjugated human CD137L protein and demonstrated that recombinant SA-hCD137L could promote the cytolytic effector function of Vγ9Vδ2-T cells against influenza-infected MDMs by promoting the release of cytolytic granules (containing perforin and granzyme B) and an antiviral cytokine (IFN-γ) from Vγ9Vδ2-T cells, suggesting that SA-hCD137L offers a compelling alternative to anti-CD137 agonistic antibody-mediated stimulation for influenza immunotherapy.The expression of CD137 correlates well with effective antiviral immune responses; however, anti-CD137 agonistic antibodies can induce a variety of pathologies that may limit their utility in patients.12 it was not possible to use a conventional mouse or ferret model for the study of human Vγ9Vδ2-T cells. In our previous studies, we successfully established humanized mice containing functional human T and B cells, including a percentage of Vγ9Vδ2-T cells in the peripheral blood similar to that seen in humans.32 Moreover, we found that influenza viruses could efficiently replicate and cause pathology in these mice, indicating that the humanized mice established by us are suitable for the study of human Vγ9Vδ2-T cell immune responses to influenza viruses. Using humanized mice, we further showed the synergistic effect of PAM and recombinant SA-hCD137L against influenza virus is mediated by boosting human Vγ9Vδ2-T cell immunity.Since murine T cells do not express a homolog of the Vγ9Vδ2-TCR,43 An effort to avoid this exhaustion was recently made by another research group, who provided vitamin C during the ex vivo expansion of Vγ9Vδ2-T cells.44 Here, we further demonstrated that activation of the CD137/CD137L pathway could maintain the survival of Vγ9Vδ2-T cells. The strategy of targeting CD137 costimulation may provide a new solution to avoid Vγ9Vδ2-T cell exhaustion and improve γδ-T cell-based immunotherapy.Adoptive transfer requires the expansion and activation of autologous Vγ9Vδ2-T cells ex vivo and their reinfusion into patients; this approach is becoming a popular cellular immunotherapy paradigm for patients with infectious disease or cancer. The potential to expand Vγ9Vδ2-T cells in vivo using phosphoantigens such as PAM offers a comparatively cheaper and straightforward delivery alternative. However, repeated stimulation of Vγ9Vδ2-T cells by phosphoantigens often leads to Vγ9Vδ2-T cell exhaustion.This study improves our understanding of the role of CD137 in the antiviral activity of human γδ-T cells. It also provides proof-of-concept data for a novel strategy for treating influenza virus infection by targeting CD137 to improve the antiviral activity of human γδ-T cells and increase the efficacy of γδ-T cell-based immunotherapy by using the combination of a phosphoantigen and CD137 agonist.21 Briefly, huPBMC were isolated from the buffy coats of healthy donor samples from the Hong Kong Red Cross by Ficoll-Hypaque  gradient centrifugation. The huPBMC were cultured in 10% FBS RPMI-1640 medium with the addition of PAM on day 0 and day 3 at a final concentration of 9 µg/ml. Recombinant human IL-2  was added at a final concentration of 500 IU/ml every 3 days beginning on day 3. After 14 days of culture, Vγ9Vδ2-T cells were purified by negative or positive selection with a γδ-T cell isolation kit according to the manufacturer’s instructions . Human MDMs were generated from mononuclear cells as previously described.16 In brief, adherent monocytes were seeded in 24-well plates at 5 × 105 cells/well and cultured in RPMI-1640 medium supplemented with 5% autologous serum. Then, they were allowed to differentiate into macrophages for 14 d. The purity of the monocytes, as determined by flow cytometry with an anti-CD14 monoclonal antibody, was consistently >90%.PAM-expanded Vγ9Vδ2-T cells were generated as described before.+ and CD137− Vγ9Vδ2-T cells were isolated by selection using the MACS CD137 MicroBead Kit  according to the manufacturer’s instructions. The purities of the resulting cell populations were checked routinely by flow cytometry. CD137+ and CD137− Vγ9Vδ2-T cells purities generally exceeded 95%.PAM-expanded Vγ9Vδ2-T cells were activated with an anti-γδ-TCR mAb (0.5 µg/ml) in vitro for 6 h, and then the subsets of CD137E. coli strain BL21 (DE3) as an inclusion body after induction at 37 °C for 4 h with 0.3 mM IPTG. The inclusion bodies were washed and solubilized with 8 M urea in a TBS solution. After filtering through a 0.45-μm membrane filter, the protein was purified with Ni-nitrilotriacetic acid affinity chromatography  according to the manufacturer’s instructions. The purified protein was refolded by dialysis, which gradually removed the urea. Bacterial endotoxin contaminants were removed by using DetoxiGel Endotoxin Removing Gel . The prepared recombinant SA-hCD137L protein was then filtered through a 0.2-μm membrane and quantitatively measured with the BCA Protein Assay Kit .The DNA sequence encoding core streptavidin  and the extracellular domain of human CD137L (amino acid residues 58-254) with an N-terminal 6×His tag was synthesized by Shanghai Sangon Biological Engineering Technology & Services Company Ltd. The DNA fragments were inserted into the pETH expression vector, and the recombinant SA-hCD137L protein was expressed in the 16 Viral titers were determined by daily observation of the cytopathic effect on cells infected with serial dilutions of virus stock; the median tissue culture infective dose (TCID50) was calculated according to the Reed-Muench formula. For in vitro experiments, day 14-differentiated MDMs were infected with influenza virus at a multiplicity of infection (MOI) of 2. After 1 h of viral absorption, the cells were washed with PBS to remove unabsorbed virus. Humanized mice were generated with 4- to 5-week-old male or female Rag2−/− γc−/− mice by reconstitution with whole huPBMC or Vγ9Vδ2-T cell-depleted huPBMC as we described previously.21 Four weeks after huPBMC transplantation, mice were successfully engrafted and became stable with a functional human immune system. Established humanized mice or 6- to 8-week-old Rag2−/− γc−/− mice were infected intranasally (i.n.) with the PR8 virus strain  under anesthesia. For Rag2−/− γc−/− mice, CD137+ Vγ9Vδ2-T cells, CD137− Vγ9Vδ2-T cells or whole Vγ9Vδ2-T cells (5 × 106/mouse) in 200 µl of PBS were adoptively transferred intravenously (i.v.) after infection with PR8 at the indicated time. For humanized mice, SA-hCD137L (15 µg/mouse) and PAM  were injected intraperitoneally (i.p.) at the indicated time. Mice treated with an equivalent volume of PBS were used as controls. Survival was monitored, and the infected mice were weighed daily. The lungs were collected at the indicated time for viral titer and histology assays.A mouse-adapted influenza H1N1 (A/PR/8/34) virus was cultured in Madin-Darby canine kidney cells, as described previously.+ Vγ9Vδ2-T cells, CD137− Vγ9Vδ2-T cells or whole Vγ9Vδ2-T cells  were cocultured with PR8-infected MDMs  at an E/T ratio of 10:1 for 6 h. In some experiments, neutralizing antibodies against CD137  were used to block CD137-mediated pathways, SA-hCD137L (500 ng/ml) was used to activate CD137-mediated pathways, or mouse IgG1  or PBS was used as a control. Afterward, nonadherent cells were harvested directly. Adherent cells were detached with 0.25% trypsin-EDTA. All adherent and nonadherent cells were stained with an anti-CD3 antibody to identify Vγ9Vδ2-T cells and ethidium homodimer-2  to identify dead cells. The cytotoxicity of Vγ9Vδ2-T cells against virus-infected MDMs was assessed by flow cytometry as the percentage of EthD-2+ cells in the CD3- population, as we described previously.16CD1377 cells) were labeled with 5 μM carboxyfluorescein succinimidyl ester  and then cultured as described previously to generate PAM-expanded Vγ9Vδ2-T cells. A neutralizing anti-CD137 mAb (5 μg/ml) was added to block the CD137-mediated signaling pathway, and mouse IgG1 (5 μg/ml) was used as an isotype control. On day 7, the profile of CFSE in CD3+Vγ9+ cells was detected by flow cytometry.Fresh huPBMC /M1) containing the target gene was used as a standard. The lungs from influenza virus-infected mice were harvested at the indicated time and homogenized in PBS. Total RNA was extracted with an RNeasy plus mini kit (QIAGEN) following the manufacturer’s instructions. Using the QuantiNova Probe RT-PCR Kit (QIAGEN), one-step qRT-PCR was applied to detect viral RNA with primers  and a TaqMan probe (5′[Fam]-TCAGGCC CCTCAAAGCCGAG-[BHQ-1]3′). The cycling conditions on the ABI Prism 7900HT Fast Real-Time PCR System consisted of 45 °C for 10 min and 95 °C for 5 min, followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s.Viral RNA copies in the lungs of PR8-infected mice were evaluated with a real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay by targeting the conserved matrix gene of influenza virus.31 All samples were acquired with a FACS LSR II (BD) and analyzed with FlowJo software (Tree Star).Cells were stained for surface markers with the following antibodies (BioLegend): anti-CD3 (HIT3a), anti-TCRVδ2 (B6), anti-TCRγ9 (B3), anti-CD25 (BC96), anti-CD69 (FN50), anti-NKG2D (1D11), anti-Fas (DX2), anti-FasL (NOK-1), anti-TRAIL (RIK-2), anti-CD107a (H4A3), anti-CD14 (63D3) and anti-CD137 (4B4-1). For intracellular staining, cells were fixed, permeabilized, and then stained with anti-IFN-γ (B27), anti-perforin (B-D48), anti-granzyme B (QA16A02) and anti-granzyme A (CB9) antibodies or relevant isotype control antibodies as described previously.The lungs of PR8-infected mice were collected at the indicated time, fixed with 10% formalin for 24 h and maintained in 70% ethanol. Paraffin-embedded lungs were sectioned and stained with hematoxylin and eosin.−/− γc−/− mice or humanized mice after infection. The differences in cell death, fluorescence intensity, cell percentages, cell numbers and viral titers between the control and treatment groups were analyzed by an unpaired, two-tailed Student’s t-test. The p-value of the difference in survival was determined by the Kaplan-Meier log-rank test. All these analyses were performed with GraphPad Prism software , with p-values <0.05 considered significant.Data are shown as the mean ± SEM. Multiple regression analysis was used to test the differences in the body weight changes in Rag2Supplementary Information


Scientific Reports 10.1038/s41598-020-60527-z , published online 26 February 2020.Correction to: The original version of this Article contained errors.In the original PDF version, the published date was omitted and now reads:“Published: 26 February 2020”.In addition, there were typographical errors in the Methods section, under the subheading “Experimental protocol and data acquisition”, where:“In the scan room with dimmed lights, participants were not required to keep looking at a cross mark in the center of the monitor screen, think of anything in particular, and not to sleep.”now reads:“In the scan room with dimmed lights, participants were required to keep looking at a cross mark in the center of the monitor screen, think of nothing in particular, and not to sleep.”In Table 2 where the ‘Contribution’ value for ID #7 was incorrect, the value “\” now reads “0.263”.In the Results section under the subheading “Effect of treatment with antidepressants”, where BDI and HAMD values were reversed,p = 7.29 × 10−7; and HAMD Pre = 31.3 (7.48), HAMD Post = 18.7 (12.2), paired t-test results on HAMD: p = 7.26 × 10−7).”“(Mean(SD): BDI Pre = 19.1 (5.63), BDI Post = 11.3 (5.41), paired t-test results on BDI: now reads:p = 7.26 × 10−7; and HAMD Pre = 19.1 (5.63), HAMD Post = 11.3 (5.41), paired t-test results on HAMD: p = 7.29 × 10−7).”“(Mean(SD): BDI Pre = 31.3 (7.48), BDI Post = 18.7 (12.2), paired t-test results on BDI: In the Supplementary Information file, there were missing values in Table S1c under the “Time 2 (6-8w)” column and on the “Severity of symptoms (BDI-II) “and “Observer-rated depression scale (HAMD-17)” rows. These values now read “18.7 (12.2)” and “11.3 (5.4)” respectively.These errors have now been corrected in the PDF and HTML versions of the Article, and in the accompanying Supplementary Information file.


The supramolecular structure of the compound is stabilized by a three-dimensional array of N—H⋯O and C—H⋯N hydrogen bonds and C—H⋯π interactions. 28H21N3O, the 1,2-dihydropyridine ring of the 1,2,7,8-tetrahydroisoquinoline ring system is planar as expected, while the cyclohexa-1,3-diene ring has a twist-boat conformation, with Cremer–Pople parameters QT = 0.367 (2) A, θ = 117.3 (3)° and φ = 327.3 (4)°. The dihedral angles between the best planes through the isoquinoline ring system and the three phenyl rings are 81.69 (12), 82.45 (11) and 47.36 (10)°. In the crystal, molecules are linked via N—H⋯O and C—H⋯N hydrogen bonds, forming a three-dimensional network. Furthermore, the crystal packing is dominated by C—H⋯π bonds with a strong interaction involving the phenyl H atoms. The role of the intermolecular interactions in the crystal packing was clarified using Hirshfeld surface analysis, and two-dimensional fingerprint plots indicate that the most important contributions to the crystal packing are from H⋯H (46.0%), C⋯H/H⋯C (35.1%) and N⋯H/H⋯N (10.5%) contacts.In the title compound, C The dihedral angles between the best planes through the isoquinoline ring system and the three phenyl rings  are 81.69 (12), 82.45 (11) and 47.36 (10)°, respectively. All bond lengths  and Fig. 4b) show the front and back sides of the three-dimensional Hirshfeld surface of the title compound plotted over dnorm in the range −0.4556 to 1.6207 a.u. Here, the bright-red spots appearing near O1 and N3 result from the N2—H2B⋯O1 and C7—H7B⋯N3 interactions, which play a significant role in the molecular packing of the title compound. The overall two-dimensional fingerprint plot for the title compound and those delineated into H⋯H, C⋯H/H⋯C, N⋯H/H⋯N and O⋯H/H⋯O contacts are illustrated in Fig. 5The Hirshfeld surfaces and two-dimensional fingerprint plots were calculated using et al., 2016et al., 2020S,2R)-2--1,2-diphenylethanol -4-fluoro-3-(4-methoxyphenyl)-1-oxo-2-phenyl-1,2,3,4-tetrahydroisoquinoline-4-carboxylic acid -benzyl 3-phenylcarbamoyl-1,2,3,4-tetrahydroisoquinoline-2-carboxylate -6,7-dimethoxy-1-phenyl-1,2,3,4-tetrahydroisoquinolin-3-yl]-4-phenyl-1,3-thiazole  interactions. In the crystal of CARCOQ, molecules are linked by an O—H⋯O hydrogen bond, forming chains propagating along the a-axis direction. The chains are linked by C—H⋯F hydrogen bonds, forming layers lying parallel to the ab plane. In LAQKUL, there are two independent molecules in the asymmetric unit. The heterocyclic ring assumes a twisted boat conformation and N—H⋯O interactions help to construct the three-dimensional network within the crystal packing. In AZUSOE, no classical hydrogen bonds nor π–π interactions were found in the crystal structure.The compound KUGLIK co-crystallizes with three water molecules in the asymmetric unit, which leads to the formation of intense hydrogen bonding in the crystal. In the crystal of POPYEB, molecules are packed in a herringbone manner parallel to (103) and  in acetonitrile (40 ml) was added malononitrile (5.2 mmol). The solution was stirred for 5 min at room temperature, ethylenediamine (5.2 mmol) was added and the mixture refluxed for 4 h and cooled down to room temperature. The reaction product precipitated from the reaction mixture as pale-yellow single crystals, was collected by filtration and purified by recrystallization in ethanol/water solution .To a solution of 2-acetyl-5-oxo-1H NMR : 3.2 ; 4.25 ; 6.7 ; 6.8 ; 6.9–7.7 .13C NMR : 35.17 (CH-Ar), 43.57 (CH2), 109.86 (=CH), 117.67 (=Cquat), 119.52 (CN), 125.96 (2CHarom), 126.72 (2CHarom), 126.86 (CHarom), 127.39 (CHarom), 127.99 (2CHarom), 128.66 (2CHarom), 128.89 (2CHarom), 128.89 (2CHarom), 129.21 (=Cquat), 129.56 (CHarom), 129.94 (=Cquat), 135.27 (N—Car), 139.07 (Car), 139.40 (Car.), 144.14 (=Cquat—N), 160.73 (O=Cquat—N), 167.96 (=Cquat—Ar).2 group were located in the difference-Fourier synthesis and refined isotropically with Uiso(H) = 1.2Ueq(N). All C-bound H atoms were positioned geometrically and refined using a riding model, with C—H = 0.93–0.98 Å, and with Uiso(H) = 1.2Ueq(C). Two reflections, (0 1 1) and (1 0 1), affected by the incident beam-stop and owing to poor agreement between observed and calculated intensities, nine outliers, , determined using 1593 quotients [(I+) − (I−)]/[(I+) + (I−)]  I. DOI: 10.1107/S2056989021000785/vm2245Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021000785/vm2245Isup3.cmlSupporting information file. DOI: 2058071CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Aurantiochytrium. The glyceride accounted for approximately 0.1 -0.4% of the lyophilized cells. The glyceride consisted of peaks I (85%) and II (15%). The structures of the intact and acetylated glycerides were elucidated by liquid chromatography-quadrupole time-of-flight chromatograph mass spectrometer (LC–Q/TOF) and NMR spectroscopy. The hydrate type of M-glyceride was detected as a minor component by LC–MS/MS. By 2D-NMR experiments, peaks I of the intact M-glyceride were elucidated as 1,2-didocosapentaenoyl-glyceryl-2′-oxy-3′-oxomorpholino propionic acid, and peak II was estimated 1,2-palmitoyldocosapentaenoyl- and/or 1,2-docosapentaenoylpalmitoyl-glyceryl-2′-oxy-3′-oxomorpholino propionic acid. The double bond position of docosapentaenoic acid was of the ω − 6 type (C22 = 5.ω − 6). The M-glyceride content varied by the cell cycle. The content was 0.4% of lyophilized cells at the mid logarithmic phase, and decreased to 0.1% at the mid stationary phase. When cells were grown in 1.0 µM M-glyceride-containing growth media, cell growth was stimulated to 110% of the control. With 0.1 µM acetyl M-glyceride, stimulation of 113% of the control was observed. Finding morpholine derivatives in biological components is rare, and 2-hydroxy-3-oxomorpholino propionic acid (auranic acid) is a novel morpholine derivative.A novel acidic morpholine-derivative containing glyceride (M-glyceride) was isolated from the cells of two strains of the thraustochytrid,  Aurantiochytrium has been examined extensively as a producer of high contents of squalene, docosahexaenoic acid (DHA) and pentadecanoic acids as an odd-carbon chain fatty acid4. It is well known that the major lipids of Arantiochytrium are comprised of squalene, carotenoids, triglyceride, glycolipids and phospholipids8.The thraustochytrid Aurantiochytrium sp. SYLR6#3 and NB6-3, we found a novel acidic morpholine derivative containing glyceride (M-glyceride), which consisted of 2-hydroxy-3-oxo- morpholino propionic acid, glycerol and fatty acids.During the investigation of acidic lipids from cells of 9. Nevertheless, finding morpholine derivatives from biological samples is very rare.Morpholine, a six-membered heterocyclic compound containing nitrogen and oxygen atoms, is an important moiety in many industrial and organic syntheses, and is often utilized in the field of medicinal chemistry for its advantageous physicochemical, biological, and metabolic propertiesAurantiochytrium cells and their cell growth activity.In this paper we describe the chemical structural elucidation of M-glyceride isolated from f 0.73 on TLC using chloroform/methanol/water  as the solvent. The spot on the TLC plate was negative against the molybdenum reagent10.Cells were harvested 72 h after inoculation, and the yield of M-glyceride was obtained as approximately 0.2% of the lyophilized cells. M-glyceride showed an RAurantiochytrium sp, SYLR6#3 and NB6-3.On the HPLC chromatogram of purified M-glyceride, two peaks were detected at Rt-12.54 (peak I) and 14.48 min (peak II). Peaks I and II comprised 85% and 15% of the total area, respectively , H-2  and H-3 . Furthermore, carbons at positons 1 and 2 of glycerol were esterified with fatty acids. The C-3 of glycerol was combined with a polar moiety by an ether linkage. In the 13C NMR spectrum, four quaternary carbons were detected between 160 and 180 ppm. The HMBC spectra suggested that the carbons at C-1 of DPA I (173.38 ppm) and DPA II (173.67 ppm) were assigned as esterified. The chemical shift at 177.17 ppm suggested that a carboxylic acid group was present in M-glyceride. The quaternary carbon at 169.34 ppm was suggested to be the carbonyl carbon at C-3 (> C=O) of the morpholine ring.The 7H10NO5) by an ether linkage.From the NMR results, the structure of peak I of M-glyceride was determined as a diacylglyceride combined with a morpholine moiety ] from peak I and m/z 830.5789, [C48H80NO10 ] from peak II were observed. From the major peak I, a fragment ion at m/z 574 [m/z 904–330 (C22H34O2)] was found. The liberated ion was due to the neutral loss of docosapentaenoic acid . The other fragment ions were observed at m/z 886 [loss of H2O from m/z 904], 329 [C22H34O2–H (DPA)]−, 244 [C10H15NO6–H]− and m/z 188 [C7H10NO5–H]−. In the case of peak II, fragment ions were observed at m/z 812 [loss of H2O from m/z 830], 574 [m/z 830–256 (C16H32O2)], 500 [m/z 830–330 (C22H34O2)], 329 [(C22H34O2)–H]−, and 255 [(C16H32O2)–H]−, m/z 244 [C10H15NO6–H]− and 188 [C7H10NO5–H]− were also detected. The fragment ion at m/z 255 was estimated as palmitic acid (C16 = 0). The fragment ions at m/z 244 [[C10H15NO6–H]−, 188 [C7H11NO5–H]− and 170 [C7H9NO4–H]− were found in the spectra of both peaks I and II. The results suggested that peaks I and II consisted of the same moiety. These results are summarized in Table In negative ion mode of the MS spectra, values of 7H10NO5) obtained from NMR. The MS results suggested that the morpholine moiety was C7H12NO6. The MS data of the morpholine moiety are explained as [C7H10NO5 + H2O]. This suggests that an extra H2O combined somewhere with the moiety and it exists as a hydrate. From the NMR and MS results, we considered that peaks I and II contained M-glyceride as the major component and its hydrate as the minor component  was applied to the LC–MS/MS, a large peak in the ion chromatogram of the total product ion was observed at Rt 1.225 min. In the case of the precursor ion of m/z 366 (C14H23NO10), a small broad peak was observed at Rt 1.152 min  as the minor peak (14%).To elucidate the structure of the morpholine moiety, the fatty acids in M-glyceride were substituted with an acetyl group. Acetylated M-glyceride was analysed by reverse-phase LC–Q/TOF. When the precursor ion of  348 C14H1NO9 was The LC–MS/MS fragments in positive and negative mode of both glycerides are shown in Table The formula of the fragments showed that all structures of the fragments included a morpholino propionic acid moiety.10H15NO6 (m/z 244 in negative mode and m/z 246 in positive mode), C7H11NO5 (m/z 188 in negative mode), and C7H9NO4 (m/z 170 in negative mode and m/z 172 in positive mode) were obtained from both the intact M-glyceride and acetylated M-glyceride. Therefore, the results showed that the morpholino propionic acid moiety in the intact M-glyceride was preserved in acetylated M-glyceride.As shown in Tables 1H–1H COSY and HMBC spectroscopy revealed the spin systems of glycerol, morpholine and propionic acid  to H-6 (δ 3.75 ppm) of the morpholine unit was determined. On the morpholine ring unit, the chemical shift of H-6 (δ 3.75 ppm) and C-6 (δ 66.91 ppm) suggested that C-6 was connected to an oxygen atom. Additionally, the chemical shifts of H-5 (δ 3.18 ppm) and C-5 (δ 46.16 ppm) suggested that C-5 was connected to a nitrogen atom. Furthermore C-3 of the morpholine ring unit correlated with H-2 and H-5 of the ring in the HMBC spectrum. An HMBC correlation was also observed between C-2 and H-6 of the morpholine ring. From these results, the morpholine ring was elucidated.On the propionic acid unit of the morpholine moiety, the relation of the connectivity from H-2 (δ 2.45 ppm) to H-3 (δ 3.48 ppm) was observed in the COSY spectrum, and the correlations of C-1 (δ 177.42 ppm) with H-2 and H-3 of the propionic acid unit were found in the HMBC spectrum. The HMBC spectrum also showed that H-3 of the propionic acid unit correlated with C-3 and C-5 of the morpholine ring unit.1), whereas the structure of the extra H2O containing acetylated M-glyceride (hydrate) was still unclear.From these results, the structure of acetylated M-glyceride was elucidated as 1,2-diacetylglyceryl-2’-oxy-3’-oxomorpholino propionic acid .The intact M-glyceride consisted of two fatty acid molecules. Peak I, as the major peak Fig.  containe1H- and 13C-NMR spectra (Table H2-CH = (δ 2.07 ppm), = CH-CH2-CH = (δ 2.85 ppm) and –CH = CH-(δ 5.38 ppm), were also assigned.Regarding the chemical shifts of DPA in peak I of the intact glyceride in the ra Table , C-1 of 411, and the formed alkanoic acid was identified as a methyl ester derivative by GC. A peak was detected at Rt 4.763 min. This Rt agreed well with authentic methyl hexanoate. From these results, DPA was identified as ω − 6 docosapentaenoic acid. The fatty acid of peak II was also identified as ω − 6.DPA has two isomers of the ω − 3 and ω − 6 types. To confirm the isomers, the fatty acids of peaks I and II were oxidized using KMnO4/KIO22 = 5, ω − 6) glyceryl-2′-oxy-3′-oxomorpholino propionic acid , and the minor one was estimated as 1,2-palmitoyl-docosapentaenoyl- and/or 1,2-docosapentaenoyl-palmitoyl-gryceryl-2′-oxy-3′-oxomorpholino propionic acid.From the above results, the major M-glyceride peak I was elucidated as 1,2-didocosapentaenoyl- of acetylated M-glyceride hydrate was obtained from MS/MS spectra 2] ought to have appeared at 100 ppm, as shown for the chemical shift of C-2  of the morpholine moiety. However, the chemical shift of C-3 appeared at 163.91 ppm  of the mitotic period. M-glyceride may play an important role in cell division. Most likely, the concentration of endogenous M-glyceride is sufficient for cell division, and exogenous M-glyceride is not necessary. After the logarithmic phase, the concentration of endogenous M-glyceride rapidly decreased, and cell division slows down. At this time, exogenous M-glyceride may be incorporated and utilized for cell growth.M-glyceride is a negative charged biosurfactant. At a high concentration (100 or 1000 µM), cell growth was inhibited after 24 h of cultivation. This inhibition may be related to the surfactant action of M-glyceride, because the inhibition induced by acetylated M-glyceride was not observed clearly.12. Polygonapholine, a novel alkaloid, was isolated from Polygonatum altelobatum13. Furthermore, an anti-tumor antibiotic morpholine derivative was isolated from Streptomyces globisporus14. A morpholine derivative, syn-3-isopropyl-6-(4-methoxybenzyl)-4-methylmorpholine-2,5-dione, was isolated from the Thai Sea hare, Bursatella leachii15. The above findings on morpholine moieties in secondary metabolites have been considered rare cases. Our finding is unique because the morpholine moiety is a component of diacylglyceride as a lipid metabolite. The elucidated polar moiety, 2-hydroxy-3-oxomorpholino propionic acid (auranic acid) is a novel morpholine derivative.A few reports on morpholine derivatives from natural products have been described. Chelonins were isolated from a marine sponge that had antimicrobial and anti-inflammatory activitiesAurantiochytrium and other organisms.The morpholine derivative -containing glyceride possibly plays an important role in the life cycle of 19. Morpholine derivatives isolated from natural products as in this study, may contribute to the development of a new medicinal drug.Morpholine derivatives have been synthesized for medicinal drugs and their biological activities have been examinedDEAE- Sephadex-A25 was obtained from GE Healthcare UK Ltd . Yeast extract and tryptone were purchased from Becton, Dickinson and Company . Sea salt was obtained from Red Sea Salt USA . All other chemicals and solvents were of analytical grade.Aurantiochytrium sp SYLR6#3 and NB6-3 isolated from seawater in the Okinawa Islands, Japan, were purchased from OP BIO Co. .The seed culture medium contained 1% tryptone, 0.2% yeast extract and 2% glucose, which were dissolved in 1.2% sea salt solution.Mass-culture was performed in 5 L air-lift bioreactors  equipped with a pH controller. Mixing of the medium was performed by air bubbling. The bioreactors were set up in a clean booth, which were maintained at 25 ± 1.2 °C. The mass-culture medium contained 3.6% glucose, 0.5% sodium glutamate, 1.0% tryptone, 0.2% yeast extract and 1.0% sea salt. Air was supplied at 1.2 vvm. To make bubbles, air was passed through a ceramic sparger. Cells were grown in the medium at pH 7.4, controlled by the addition of 1.0 M NaOH, and were harvested at 72 h after inoculation. For the 72h culture, the glucose content in the medium reached below 0.2%. Harvested cells were lyophilized and stored at − 25 °C.Lipids were extracted with chloroform (C)/methanol (M) 2:1(v/v) from 10 g of lyophilized cell materials. To the extract, 0.2 volumes of 0.9% NaCl solution was added. After centrifugation, the lower phase was separated and evaporated using a rotary evaporator under reduced pressure. The remaining residue was dissolved with C, and fractionated by silica gel chromatography , eluted with 100 mL of C, CM  and CM (1:4). The phospholipid (PL) fraction was recovered from CM (1:4). After removing the solvent, the PL fraction was resuspended in C/M/ water (W) . The suspension was applied to DEAE-Sephadex-25 , and 100 mL of C/M/W  was passed through the column, followed by 100 mL of C/M/0.2 M ammonium formate . The acidic PL fraction was eluted with an ammonium salt containing solvent. The eluate was evaporated under reduced pressure. The M-glyceride and acidic PL containing residue was suspended in 0.1% phosphoric acid containing water. M-glyceride was extracted from the suspension with n-hexane.f of 0.73. Under UV light, the M-glyceride band was scraped off the plates and eluted with C/M . M-glyceride was re-chromatographed on silica gel plates using the same solvent system. The purified M-glyceride was utilized for further structural analysis.To purify M-glyceride from the extract, the extract was applied to TLC silica gel plates containing a fluorescent indicator and developed with C/M/W  as the developing solvent. M-glyceride migrated at an R20. After hydrolysis, the pH of the hydrolysate was adjusted to 4.0 with 1.5 M HCl. The fatty acids liberated from M-glyceride were extracted with n-hexane. After neutralization with 1.0 M NaOH, the aqueous phase was evaporated to dryness under a stream of N2. The remaining residue was subjected to acetylation using pyridine/acetic anhydride  at 25 °C overnight. After the reaction, the solution was dried under a stream of N2. The remaining residue was suspended in n-hexane, spotted on TLC plates and developed with CMW  as the solvent. The acetylated M-glyceride on TLC was detected by charring after spraying with 20% H2SO4. The Rf value of the acetylated lipid was found to be 0.61. The acetylated M-glyceride was scraped off from the plates and extracted with CM . As a result, a colourless pasty material was obtained after drying under a stream of N2, which was utilized for NMR and LC–MS/MS analyses.M-glyceride was subjected to mild alkaline hydrolysis at 25 °C for 1 h in methanolic 1.5 M NaOHM-glyceride was further purified using Shimadzu Prominence HPLC consisting of pumps (LC-20AD), a controller, a degasser, a column oven, a PDA detector and a workstation. The HPLC conditions were as follows: column, Wakosil 5 NH4 column (4.6 × 150 mm); column temperature, 40 °C; mobile phase, acetonitrile/methanol/0.2% trimethylamine in water adjusted to pH 4.0 with phosphoric acid ; and flow rate, 1.0 mL/min. Peaks were detected at 205 nm.3-methanol at 70 °C for 20 min. Fatty acid methyl esters were analysed using GC-FID (Shimadzu GC-2025) with a DB-23 column . The GC operating conditions were as follows: column temperature, 50 °C held for 1 min, increased to 175 °C (at a rate of 25 °C /min), then increased to 230 °C (at a rate of 4 °C/min), and held for 5 min; FID port temperature, 250 °C; carrier gas (He) flow rate, 2.06 mL/min; FID hydrogen gas flow rate, 40 mL/min; and air flow rate, 450 mL/min.Fatty acids of M-glyceride were converted to methyl esters using 14% BF1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were recorded using a JEOL JNM-ECA500 spectrometer. CDCl3 or CDCl3/CD3OD  was used as the solvent.Nexera UHPLC and aquadrupole time-of-flight mass spectrometer . LC conditions were as follows; column, Kinetex C8  Phenomenex, USA; solvent, isocratic, 30% of 20 mM ammonium formate-containing and 70% acetonitrile/isopropanol ; flow rate, 0.4 mL/min; and column temperature, 40 °C. MS conditions were as follows: ionization mode, ESI positive/negative; capillary voltage, 4.5 kV (positive)/− 3.5 kV (negative); drying gas, 10 L/min; nebulizer gas, 2.0 L/min, heating gas, 10 L/min; interface temperature, 300 °C.MS and MS/MS spectra were recorded on an LC/Q-TOF system consisting of a A. sp. were cultured with growth medium  containing various concentrations (1.0–1000 µM) M-glyceride or (0.1–100 µM) acetylated M-glyceride in 16-well plates at 25 °C for 24 or 48 h. Cell concentrations were determined using the photoelectric colourimeter at 650 nm and were expressed as the absorbance of the cell suspension.Cells of


Toll-like receptor 4 (TLR4) is a crucial receptor in neuroinflammation and apoptotic neuronal death, and increasing evidences indicated that β2-microglobulin (B2M) is thought to be a major contributor to age-related cognitive decline. In present study, we designed to investigate the effects of TLR4 on B2M-induced age-related cognitive decline. Wild-type (WT) C57BL/6, TLR4 knockout (TLR4 -KO) mice and hippocampal neurons from the two type mice were respectively divided into two groups: (1) Veh group; (2) B2M-treated group. The behavioral responses of mice were measured using Morris Water Maze. Hippocampal neurogenesis and neuronal damage, inflammatory response, apoptosis, synaptic proteins and neurotrophic factors, and TLR4/MyD88/NF-κB signaling pathway proteins were examined using molecular biological or histopathological methods. The results showed that WT mice received B2M in the DG exhibited age-related cognitive declines, increased TLR4 mRNA expression and high levels of interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and apoptotic neuronal death in the hippocampus, which were partially attenuated in TLR4-KO mice. Moreover, in absence of TLR4, B2M treatment improved hippocampus neurogenesis and increased synaptic related proteins. Our cell experiments further demonstrated that deletion of TLR4 could significantly increase synaptic related protein, decrease neuroinflammatory fators, inhibited apoptotic neuronal death, and regulated MyD88/NF-κB signal pathway after B2M treatment. In summary, our results support the TLR4 contributes to B2M-induced age-related cognitive decline due to neuroinflammation and apoptosis through TLR4/MyD88/NF-κB signaling pathway via a modulation of hippocampal neurogenesis and synaptic function. This may provide an important neuroprotective mechanism for improving age-related cognitive decline. Literatures have noted that advanced age is the highest risk factor for physiological decline in both physically and psychologically, including decreased neurogenesis and cognitive impairment –3. SerioHeterochronic parabiosis evidences have demonstrated an age-dependent bi-directionality in the influence of the systemic environment indicating pro-aging factors in old blood drive aging. It has also been proposed that alleviating the effect of pro-aging factors may provide an effective approach to rejuvenate aging phenotypes , 6. Therβ2-microglobulin (B2M), a light chain of major histocompatibility complex class I (MHC I) , is a ciToll-like receptor 4 (TLR4) is a type of pattern recognition receptor, and is involved in innate and adaptive immunity by producing pro-inflammation cytokines, chemokines and apoptosis  . StudieIn this study, we hypothesized that neuroinflammation and apoptosis triggered by TLR4 may contribute to B2M-induced cognitive dysfunction. Using B2M-induced cognitive dysfunction model of wild-type (WT), TLR4 knockout (KO) mice and hippocampal neurons, we aimed to investigate the role of TLR4 in age-related learning and memory deficits. Thus, we tested the cognitive function of B2M-handled mice, and assessed the hippocampal structure changes, hippocmapal neurogenesis, synaptic protein, apotosis, as well as the expression of TLR4 mRNA and the levels of IL-1β, TNF-α, NGF and BDNF.Previous study showed that exogenous B2M injected locally in the hippocampus impairs hippocampus-dependent cognitive function in WT mice. However, it is not clear whether the same treatment affects the behavior of TLR4-KO mice. The evidence demonstrated the role of TLR4 in learning and memory function of Alzheimer’s disease (AD), we wondered if TLR4 affected B2M-induced cognition impairment. Therefore, we tested the effect of B2M, administrated in hippocampus, on cognitive behaviors in WT mice and TLR4-KO mice by Morris water maze test.p = 0.0014; day4: 34.6 ± 3.1 for B2M group vs. 21.2 ± 1.8 for Veh group, p < 0.0001) and TLR4-KO mice . Moreover, TLR4 elimination alleviated B2M-induced learning impairment .With the training for 4 consecutive days, all groups of mice improved in the ability to reach the goal platform throughout the training trials, meaning a learning process in the test. As shown in Fig. p = 0.0139; TLR4-KO mice: 1.1 ± 0.6 for B2M group vs. 3.1 ± 0.8 for Veh group, P = 0.0002; time in the target zone: WT mice: 12.9 ± 3.7 for B2M group vs. 26.7 ± 5.5 for Veh group, p < 0.0001; TLR4-KO mice: 20.3 ± 3.3 for B2M group vs. 37.5 ± 9.4 for Veh group, p = 0.0010). TLR4 elimination also alleviated B2M-induced spatial memory dysfunction as indicated by more time in the target zone .Probe trials conducted 24 h after training on day 28 after B2M treatment. As shown in Fig. p < 0.0001; day 28: 3.75 ± 0.84 for B2M group vs. 1.99 ± 0.42 for Veh group, p < 0.0001; Fig. To explore the role of TLR4 in the course of B2M-induced cognition impairment, we measured TLR4 mRNA expression in the hippocampus of the WT mice from Veh group and B2M group on day 7 and 28 after B2M treated. Here, we observed significantly higher levels of TLR4 mRNA expression at day 7 and 28 after B2M treated . TLR4 elimination caused an improvement in neurogenesis, indicated by increased BrdU/NeuN positive cells .To explore the cellular basis for the observed changes in learning and memory, we investigated the effects of B2M and TLR4 on the production of neurons in the dentate gyrus (DG) of hippocampus. A double BrdU/NeuN immunostaining was performed 22 day after BrdU administration. As show in Fig. +) and immature neurons  in hippocampus DG was examined at 7 day after B2M treated . Original full western blot images of SYN and PSD-95 were in supplementary Fig. 7 from the Additional file In cultured primary neurons, we further investigated this function, the expressions of proteins of SYN and PSD-95 were detected using Western blot Fig. a, d, e. p<0.0001; 0.34 ± 0.05 for B2M group vs. 0.08 ± 0.02 for Veh group of TLR4-KO mice, p<0.0001;TNF-α: 0.53 ± 0.11 for B2M group vs. 0.10 ± 0.05 for Veh group of WT mice, p<0.01; 0.28 ± 0.10 for B2M group vs. 0.09 ± 0.04 for Veh group of TLR4-KO mice, p<0.01; day28: IL-1β: 0.76 ± 0.10 for B2M group vs.0.11 ± 0.04 for Veh group of WT mice, p<0.0001; 0.40 ± 0.05 for B2M group vs. 0.10 ± 0.02 for Veh group of TLR4-KO mice, p<0.0001; TNF-α: 0.57 ± 0.06 for B2M group vs. 0.13 ± 0.04 for Veh group of WT mice, p<0.0001; 0.29 ± 0.07 for B2M group vs. 0.12 ± 0.03 for Veh group of TLR4-KO mice, p<0.0001), which was attenuated in TLR4-KO mice . Original full western blot images were in supplementary Fig. 5a from the Additional file We measured the levels of the inflammatory cytokines IL-1β, TNF-α in the hippocampus to investigate the role of TLR4 on inflammatory response following B2M treated Fig.  using Wep < 0.0001). Original full western blot images of IL-1β and TNF-α were in supplementary Fig. 7 from the Additional file Cultured primary neurons showed almost the same trend, as shown in Fig. p < 0.0001), and this was partially diminished in TLR4-KO mice after B2M-treated   Fig. b.Fig. 6p < 0.0001 vs. B2M in WT). Moreover, the activation of cleaved-caspase3 was significantly ameliorated in TLR4-KO group after B2M treatment. Original full western blot images of cleaved-caspase3, Bcl-2 and Bax in vivo and vitro were respectively in supplementary Fig. 6c and Fig 7 from the Additional file We also assessed whether apoptotic responses were related to TLR4 and B2M both in vivo Fig. c-e and iNext, we examined the protein levels of NGF and BDNF in hippocampus using Western blot.p < 0.001) and for genotype (p < 0.001) in the hippocampus, the levels of NGF and BDNF were decreased in WT mice at 7 days after B2M treatment , which the decrease was attenuated in TLR4-KO mice after B2M treatment . Original full western blot images were in supplementary Fig. 6f from the Additional file As shown in Fig. p < 0.0001), and TLR4-KO group showed a slight increase in the levels of NGF and BDNF . Original full western blot images of NGF and BDNF were in supplementary Fig. 7 from the Additional file To further explore the effect of B2M on cultured primary neurons, the expressions of proteins of NGF and BDNF were detected using Western blot Fig. i-k. Aftep < 0.0001), which were inhibited in TLR4-KO group after B2M-treated (p < 0.0001). These results suggest that B2M activates TLR4 signaling pathway and also imply that the regulation of B2M-induced age-related cognitive decline are probably mediated through TLR4 signaling pathway. Original full western blot images were in supplementary Fig. 8 from the Additional file To further analyze the regulatory mechanism of B2M-induced age-related cognitive decline, the expressions of the key nodes  in TLR4 signaling pathway were detected . All efforts were made to minimize animal suffering and reduce the number of animals used.8–12 weeks male WT (C57BL/6) mice and TLR4-KO  mice weighing between 20 and 25 g were purchased respectively from Model Animal Research Center of Nanjing University in Jiangsu, China. As reported, TLR4 knockout mice are viable with no obvious spatial learning deficits in morris water maze paradigm . Both the WT and TLR4-KO mice were housed in groups of 3–5 each per cage, and in a light-controlled room (12 h light and 12 h dark cycle) at a temperature of 24 ± 1 °C and a humidity of 55% ± 5% with free access to food and water. The animals were allowed to adapt to new environment for at least 7 d before being used in experiments.B2M  was injected to young 8–12 weeks) mice by bilateral stereotaxic . WT and TLR4-KO mice were respective randomly divided into two groups: (1) vehicle group (Veh), which received vehicle   in the bilateral DG of the dorsal hippocampus; (2) B2M-treated group(B2M), which received B2M  [–12 weeksHippocampal neurons were prepared from postnatal day 1 of C57/BL and TLR4-KO mice under sterile conditions as reported by Brewer et al.  with furCarrier free forms of human recombinant B2M (Vendor) were dissolved in PBS and added to 3 ml cell culture medium (1 μg/ml) following cell plating. We changed half of the medium with 1.5 ml cell culture medium contained 1μg/ml B2M every other day, the B2M stayed in the cell cultures for 48 h each time till the end of the 8th day and the total dosage of B2M was 7.5 μg. Neurons were respective randomly divided into two groups: (1) vehicle group (Veh), which exposed to vehicle (PBS); (2) B2M-treated group(B2M), which exposed to soluble B2M [For the B2M or vehicle treatment, all of the WT and TLR4-KO mice were anesthetized with 10% chloralic hydras  and placed in stereotaxic frame. B2M or vehicle was injected bilaterally into the DG of the dorsal hippocampus using the following coordinates: AP (anterior to posterior): − 2.0 mm, ML : ±1.5 mm, DV : − 2.60 mm. To limit reflux along the injection track, the infusion needle was left in position for 8 min, slowly pulled out halfway and kept in position for an additional 2 min.To label newborn cells, all mice received 5-bromo-2′ deoxyuridien  intraperitoneally once daily during the first six days after B2M or vehicle treatment. BrdU-labeled cells in the hippocampus were examined at 24 h or 22 days after the last BrdU injection.Morris water maze (MWM) was conducted 24 day after the B2M treatment as previously described . The cirn = 5 eyes/E-Reeler,, and resuspended in 10 μL fresh available RNAse-free water. After homogenization, total RNA was extracted using TRIzol reagent . The cDNA was synthesized using PrimeScript™RT reagent kit . For quantitative real-time PCR reaction, the SYBR Premix Ex Taq™ reagent kit  was used in StepOne™ Real-Time PCR detection system . The PCR condition was pre-denaturation at 95 °C for 60 s, followed by 40 cycles of nannealing reaction at 95 °C for 15 s, and extension 60 °C for 20 s. β-actin was used as endogenous reference gene for normalizing the quantities of TLR4 gene expression in animals, and TLR4, MyD 88 and NF-κB 65 gene expression in cell.At 7 or 28 day after B2M or vehicle treatment, 6 mice in each group of WT mice were anesthetized with chloralic hydras and the hippocampus tissues were rapidly collected and stored at − 80 °C for processing. In vitra expereiment, according to the TRIfast standard procedure (EuroClone), Total RNA was obtained from each single cell population of The sequences of the primers used in this this study were as follows:TLR4 forward: 5′-ACACTTTATTCAGAGCCGTTGGT-3′;reverse: 5′-CAGGTCCAAGTTGCCGTTTC-3′.MyD 88 forward: 5′-GGCATCTGCATATGTGTGTT-3′;reverse: 5′- CCCAGGCTGACCTTAAACTA-3′.NF-κB 65 forward: 5′- ACGACATTGAGGTTCGGTTC-3′;reverse: 5′- ATCTTGTGATAGGGCGGTGT-3′.β-actin forward: 5′-CTGAGAGGGAAATCGTGCGT-3′;reverse: 5′-CCACAGGATTCCATACCCAAGA-3′.Four mice in each group were humanely sacrificed at 24 h or 22 days after the BrdU injection. Mice were anesthetized with chloralic hydras and perfused transcardially after a thoracotomy with 100 ml of ice-cold saline, followed by 4% paraformaldehyde  at 4 °C. The fixed brain was rapidly removed, postfixed in 4% paraformaldehyde, embedded, and sectioned for immuostaining. 4 mice per group and 15 sections per mouse were analyzed, during section procedure, we choose every sixth sections mounting on slide for further analysis. For staining of BrdU-positive cells, sections were incubated with primary antibody  and then biotin-conjugated secondary antibody . In order to estimate the total numbers of immature neurons, sections were incubated with primary antibody  and then biotin-conjugated secondary antibody . For BrdU-NeuN co-labeling, sections were multi-stained by incubation with primary antibodies  and then secondary antibodies . Images were acquired using a fluorescence microscope  equipped with an imaging system.The total protein samples from the hippocampal tissues and hippocampal neurons were homogenized using RIPA lysis buffer  containing protease and phosphatase inhibitors. The lysates were collected and then centrifuged at 13000 g at 4 °C for 5 min. The resulting supernatants were collected, and the protein concentrations were determined using a bicinchoninic acid protein assay . Sample protein (40 μg) were mixed with equal volumes of 5 × sodium dodecyl sulfate protein gel loading solution , boiled for 5 min. Aliquots of each sample (10 μl containing 40 μg protein) were loaded on 9–12% polyacrylamide gels and separated by electrophoresis for 90 min at 120 V. Separated proteins were transferred to polyvinylidene fluoride membrane  for 1.5 h at 300 mA. Nonspecific binding was blocked for 1 h at 37 °C with 5% nonfat dry milk in Tris-buffered saline Tween . Membranes were incubated overnight at 4 °C with the following antibodies: rabbit anti-NGF , rabbit anti-BDNF , rabbit anti-synaptophysin , rabbit anti-PSD-95 , anti-IL-1β  and rabbit anti-TNF-α , rabbit anti-Bcl-2 , rabbit anti-Bax , rabbit anti-cleaved-Caspase-3 , rabbit Anti-MyD88, rabbit anti-NF-κB p65 . The membranes were washed 3 times with TBST at 37 °C for 15 min and then incubated with secondary antibodies . Blots were developed using the enhanced chemiluminescence technique. Band densities were quantified using AlphaEaseFC software . β-actin  was blotted on the same membrance as a loading control. The quantified values are expressed as a percentage to β-actin intensity.n = 3 per group) were deeply anesthetized and cardiac perfused with pre-cold heparinized-normal saline and 4% paraformaldehyde. After postfixed in the same fixative solution, brain samples were dehydrated, and then cryosectioned at a thickness of 20 μm. Every sixth sections through the longetutive hippocampus were selected for further study. Commercial InSitu Cell Death Detection Kit  was employed to detect neuronal apoptosis according to the manufacturer’s instructions [At 7 day after B2M or vehicle treatment, mice . The data from Q-PCR in WT mice were analyzed using one-way ANOVAs with post-hoc pairwise comparisons, the data from Bcl-2/Bax ratio were analyzed using t tests. The other data were analyzed using two-way ANOVAs used Tukey’s multiple comparisons test. The results were expressed as mean ± standard deviation. Additional file 1: This document contains additional information on the original full western blot images from all the samples.


N2,N3-bis(quinolin-8-yl)pyrazine-2,3-dicarboxamide, with Cu(ClO4)2 in acetonitrile a binuclear copper complex was formed. Both copper atoms are CuN5 coordinate, and while one copper atom has a perfect square-pyramidal coordination sphere the other has a distorted shape.On reaction of the new pyrazine dicarboxamide ligand,  N2,N3-bis(quinolin-8-yl)pyrazine-2,3-dicarboxamide (2L1H), C24H16N6O2, has a twisted conformation with the outer quinoline groups being inclined to the central pyrazine ring by 9.00 (6) and 78.67 (5)°, and by 79.94 (4)° to each other. In the crystal, molecules are linked by C—H⋯O hydrogen bonds, forming layers parallel to the (102L1H with Cu(ClO4)2 in acetonitrile leads to the formation of the binuclear complex, [μ-(3-{hydroxy[(quinolin-8-yl)imino]methyl}pyrazin-2-yl)[(quinolin-8-yl)imino]methanolato]bis[diacetonitrilecopper(II)] tris(perchlorate) acetonitrile disolvate, [Cu2(C24H15N6O2)(CH3CN)4](ClO4)3·2CH3CN or [Cu2(HL1−)(CH3CN)4](ClO4)3·2CH3CN (I). In the cation of complex I, the ligand coordinates to the copper(II) atoms in a bis-tridentate fashion. A resonance-assisted O—H⋯O hydrogen bond is present in the ligand; the position of this H atom was located in a difference-Fourier map. Both copper(II) atoms are fivefold coordinate, being ligated by three N atoms of the ligand and by the N atoms of two acetonitrile molecules. The first copper atom has a perfect square-pyramidal geometry while the second copper atom has a distorted shape. In the crystal, the cation and perchlorate anions are linked by a number of C—H⋯O hydrogen bonds, forming a supramolecular three-dimensional structure.The title pyrazine dicarboxamide ligand,  N2,N3-bis(quinolin-8-yl)pyrazine-2,3-dicarboxamide (2L1H), is very similar to ligand 2N-[methyl]-3N-(pyridin-2-ylmethyl)pyrazine-2,3-dicarboxamide (2L2H), for which two polymorphs have been reported 2 2 and NiCl2 , and of a binuclear copper complex, I, which was synthesized by the reaction of 2L1H with copper perchlorate using acetonitrile as solvent. The various intermolecular contacts in the crystal of 2L1H have been studied by Hirshfeld surface analysis.The title ligand, 2L1H is illustrated in Fig. 1N is involved in two intramolecular N—H⋯N contacts  ring motifs 2·2H2O, the bond lengths and angles involving the two amide moieties  to 2.054 (4) Å, while the apical Cu—N21 bond length is 2.137 (4) Å. The ligand is essentially planar with the quinoline ring systems (involving atoms N4 and N6) being inclined to the central pyrazine ring by 1.78 (17) and 1.80 (17)°, respectively, and by 2.65 (13)° to each other.The asymmetric unit of compound on Fig. 2, the lig2L1H, molecules are linked by two pairs of C—H⋯O hydrogen bonds (C12—H12⋯O2ii and C18—H18⋯O2iii), each involving inversion-related molecules, forming chains of loops propagating along the . The second offset π–π interaction involves inversion-related N6/C16–C24 (centroid Cg3) quinoline ring systems with Cg3⋯Cg3ii = 3.6526 (8) Å, α = 0.00 (4)°, β = 24.1°, γ = 24.1°, interplanar distance = 3.3333 (4) Å, with an offset of 1.494 Å .In the crystal of n Table 1, so formre Fig. 5. The firI, the cations are arranged in layers parallel to the (012) plane. They are linked via the perchlorate anions by a number of C—H⋯O hydrogen bonds , C⋯H/H⋯C at 24.1% , O⋯H/H⋯O at 12.2%  and N⋯H/H⋯N at 11.8%  contacts. The contributions of the C⋯N  and C⋯C  contacts are 7.7 and 6.7%, respectively.The two-dimensional fingerprint plots for 1% Fig. 9c, O⋯H/H2% Fig. 9d and N⋯8% Fig. 9e contac⋯N Fig. 9f and C⋯⋯C Fig. 9g contacet al., 2016N-(quinolin-8-yl)pyrazine-2-carboxamide pyrazine-2-carboxamidato]copper(II) monohydrate pyrazine-2-carboxamide]tetracopper(II) methanol solvate .A search of the Cambridge Structural Database :-di(quinolin-8-yl)pyrazine-2,3-dicarboxamide  was added to a solution of pyrazine-2,3-dicarboxylic acid  and 1,1′-carbonyldiimidazole  in 180 ml of DMF (anhydride) in a two-necked flask (500 ml). The solution was mixed for 15 min at room temperature and then heated gradually for 1 h and then refluxed for 7 h. The reaction mixture was then cooled and added directly to a column , and eluted with DMF. After evaporation of the solvent the solid obtained was refluxed in 80 ml of ethanol for 10 min and then filtered. The brown–yellow solid obtained was recrystallized from DMF and on slow evaporation of the solvent pale-yellow rod-like crystals of 2L1H were obtained . 1H NMR : 11.47 ; 9.04 ; 8.95 ; 8.82 ; 8.47 ; 7.77 ; 7.66 . IR : 3350 (s), 3300 (s), 1678 (vs), 1560 (vs), 1530 (vs), 1520 (vs), 1488 (vs), 1465 (s), 1427 (vs), 1385 (s), 1326 (s), 1151 (s), 1109 (s), 919 (s), 829 (vs), 792 (vs), 752 (s), 652 (s), 607 (s). Analysis. for C24H16N6O2 (Mr = 420.43 g mol−1) calculated (%) C: 68.56, H: 3.84, N: 19.99; found (%) C: 68.70, H: 3.92, N: 20.40.2(HL−)(CH3CN)4]Synthesis of complex  and wR(F2) are 0.0558 and 0.1328. The CIF, including the HKL file, has been deposited with the Cambridge Structural Database  = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms. For complex I, a resonance assisted O2—H2O⋯O1 hydrogen bond  H2L1, I, Global. DOI: 10.1107/S2056989020001838/zl2771H2L1sup2.hklStructure factors: contains datablock(s) H2L1. DOI: 10.1107/S2056989020001838/zl2771Isup3.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020001838/zl2771sup4.pdfCSD search. DOI: 10.1107/S2056989020001838/zl2771sup5.cifCrystal structure: contains datablock(s) II. DOI: 1982863, 1982862CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Low concentrations of Aβ (1 μM) induced RhoA-mediated Ser422 phosphorylation of Tau protein (p-Ser422 Tau), but reduced the expression of ATP citrate lyase (ACL) in the HT22 hippocampal neuronal cell line. In contrast, high concentrations of Aβ (10 μM) along with high levels of superoxide production remarkably attenuated accumulation of p-Ser422 Tau, but augmented ACL expression and activated sterol regulatory element-binding protein 1 (SREBP1), leading to cellular senescence. Notably, a high concentration of Aβ (10 μM) induced nuclear localization of p-Tyr42 Rho, which positively regulated NAD kinase (NADK) expression by binding to the NADK promoter. Furthermore, severe AD patient brain showed high p-Tyr42 Rho levels. Collectively, our findings indicate that both high and low concentrations of Aβ are detrimental to neurons via distinct two p-Tyr42 RhoA-mediated signalling pathways in Ser422 phosphorylation of Tau and ACL expression.Both the accumulation of Amyloid-β (Aβ) in plaques and phosphorylation of Tau protein (p-Tau) in neurofibrillary tangles have been identified as two major symptomatic features of Alzheimer's disease (AD). Despite of critical role of Aβ and p-Tau in AD progress, the interconnection of signalling pathways that Aβ induces p-Tau remains elusive. Herein, we observed that a popular AD model mouse (APP/PS1) and Aβ-injected mouse showed an increase in   •Aβ stimulates at least two signalling pathways depending on its concentrations.•p-Tyr42 RhoA is critical for the two signalling pathways.•p-Ser422 Tau.Low concentration of Aβ induces •High concentration of Aβ induces ACL expression along with highly producing superoxide.•p-Tyr42 RhoA translocalizes to nucleus, where it regulates expression of NAD kinase (NADK).In particular,  The major pathological features of AD include extracellular senile plaques, consisting of amyloid-β (Aβ) peptide and intracellular neurofibrillary tangles (NFTs) consisting of aggregated hyperphosphorylated Tau protein. Notably, the accumulation of oligomeric and fibrillary Aβ contributes to hyperphosphorylation of the Tau protein through a series of neuronal signal transduction events . HyperphTau is a microtubule (MT) associated protein and involved in the stabilization of axonal MT. The abnormal phosphorylation of Tau releases itself from MT, promoting disassembly and disruption of MT, leading to impairment of axonal MT function and ultimately to neuronal cell death. Tau proteins can be phosphorylated on at least 30 sites. Soluble Tau has a low ratio of phosphate/Tau, whereas paired helical filament Tau (PHF-Tau) has a much higher ratio of phosphate/Tau in AD brains .p-Tyr216 GSK-3β is an active form, while p-Ser9 and p-Ser389 (in mouse or p-Thr390 in human) of GSK-3β are inactive forms. In fact, p-Tyr216 GSK-3β was found in the brain of AD patients  were bred with female C57BL/6 mice and maintained under constant temperature, humidity, and 12 h-light/12 h-dark cycle. C57BL/6 mice were purchased from Samtako (Samtako Inc. South Korea). Heterozygous APP/PS1 mice were genotyped by primers for PSEN1  and APP (377 bp). APP/PS1 male mice of 4–5 generation without apparent seizure were used. A few amyloid plaques were started to develop in the cerebral cortex at the age of 4–6 months of APP/PS1 transgenic mice by Thioflavin S (ThS) staining. The experiments were performed on wild-type and APP/PS1 (C57BL/6 mice) at 9-months-old , 12-months-old , and 16-months-old . In addition, C57BL/6J mice  were used for Aβ injection to hippocampal region.2.4All mice were anesthetized with avertin and were perfused with PBS followed by 4% paraformaldehyde (PFA). The brains were post-fixed in 4% PFA at 4 °C overnight and placed in 30% sucrose before freezing. Serial 10-μm coronal sections of the brain were collected for immunohistochemistry. The sections were blocked with blocking buffer (4% BSA and 0.3% Triton X-100 in DPBS) for 1 h at room temperature and then incubated with primary antibodies at 4 °C overnight. Primary antibodies were diluted as follows; anti-phospho RhoA (Y42) , anti-NeuN , anti-MAP2 , anti-6E10 , anti-GFP , anti-VGLUT1, anti-AIF/Iba-1 , anti-GFAP  and anti-TAU (phospho S422) . The tissues were washed five times with PBST (0.1% Tween 20) and then, incubated in secondary antibodies for 2 h at room temperature before mounting with vectashield . Immunofluorescence images were acquired using a LSM 880 confocal microscope (Carl Zeiss) and then and then confocal images were taken by Z-stack images with the same intervals. For CA1 and SLM region, image stacks were obtained using a 20x objective by a 1024× 1024 pixel and the acquired images were represented with Fiji program . Generally, it is well known that anti- MAP-2, NeuN, anti-GFAP, and anti-AIF/Iba-1 are specific marker for neuron, astrocyte and microglia in previous articles. In addition, we have tried to confirm that the pattern of immunostaining in various primary neurons, and glia as well as mouse brain tissues were similar with previous articles.2.5p-Tyr42 Rho antibody was produced by Young-In Frontier . Briefly, p-Tyr42 Rho peptide (epitope peptide T37VFEN(phospho-)Y42VADIE47) was synthesized using phospho-Tyr42 precursor. Fluorenylmethyloxycarbonyl (Fmoc)-Tyr(PO(Nme2)2)-OH was used as a precursor amino acid derivative to protect phosphate of Tyr42 from reaction and the protective groups (N(Me2)2) were removed after synthesis of peptide containing p-Tyr42. The peptide was purified by using C18 column and confirmed with Mass analysis. The peptide was conjugated to BSA, which was injected into rabbit to produce polyclonal anti-p-Tyr42 Rho antibodies. The serum containing antibody was purified through three steps: proteinA-bead, non-phospho peptide-bead to exclude dephospho-Rho antibody, and then p-Tyr42 peptide-beads were exploited to purify specific p-Tyr42 Rho GTPase antibody. Polyclonal anti-phospho Tytr42 Rho antibody revealed the specificity for p-Tyr42 RhoA , and incubated with rabbit polyclonal antibody to p-Y42 RhoA (1:200 dilution) and GSK3β-Y216 (1:200 dilution) for 24 h. After washing three times, the slides were processed with Vector ABC Kit . The immunoreactive signals were developed with DAB chromogen .Paraffin-embedded tissues were sectioned in a coronal plane at 20 μm. The tissue sections were rehydrated, blocked with blocking solution  and then mounted. The images were analysed under a bright field microscope.Endogenous alkaline phosphatase was blocked using 3% H2.155 cells/well). After treating with appropriate stimulation, cells were fixed by 4% paraformaldehyde at room temperature for 15 min, stained with Coomassie Brilliant Blue R-250 (C2006) for 5 min, washed 3 times with PBS. Three images (30–50 cells/each) at different locations in each well were acquired on a TMS-F #211128 microscope (Nikon) equipped with a Nikon Digital D5100 camera. Each experiment was done 3 times. Mean neurite outgrowth length or cell size was quantified in Photoshop cc2018 software (Adobe Inc.) by applying a grid to the pictures and counting intersections of neurites with the grid lines and total cell bodies and calculating the ratio thereof.Cells were seeded either in 6-well dishes (4 × 102.16t-test (p*<0.05).The western blotting of protein bands shown and statistical significance was based on analysis done with Photoshop cc2018 software (Adobe Inc.) and Prism 8.0 software (GraphPad), respectively. Generally, the data were shown as the means ± SE of at least three independent experiments with one- or two-way ANOVA analysis; if not, the fold number of protein band on western blotting was denoted. Values of the cell image data are means of three independent experiments (30–50 cells/image and 3 images/experiment) ± SE, one- or two-way ANOVA. The fluorescence images of brain tissues were quantified from two confocal images/sample and two samples/mouse using 3 male mice and statistical analysis of significance was based on two-way ANOVA analysis . Transcriptome of human brain samples were analysed by unaired, two-tailed 33.1p-Tyr42 RhoA, leading to NF-κB activation and cancer cell proliferation. In addition, we developed a novel antibody to recognize the p-Tyr42 residue of Rho GTPase [p-Tyr42 Rho GTPase in HT22 cells  and CA2 regions by 5 μl of 10 and 100 μM Aβ (p-Tyr42 Rho in the CA1 region of hippocampus in 12 month-old APP/PS1 transgenic mice brain. P-Tyr42 Rho found to be in CA1 and stratum lacunosum moleculare (SLM) layers of neuron  nor ionized calcium-binding molecule 1 , suggesting that p-Tyr42 Rho does not generally exist in glial cells , a synaptic vesicle marker-positive neuron associated with Aβ plaque in the APP/PS1 mouse brain  in HT22 cells (p-Ser422 Tau) levels in HT22 cell, and quantified p-Ser422 Tau levels. In contrast, NGF reduced p-Ser422 Tau level , but was inhibited by Tat-C3 and Y27632  as well as ACL, while increased p-Tyr216 GSK-3β (active), p-Tyr416 Src (active), p-Ser422 Tau in HT22 cells (p-Thr180/Tyr182 p38 (active), mSREBP1, and p-Ser389 GSK-3β (inactive), while attenuated p-Tyr216 GSK-3β (active), p-Tyr416 Src (active), and p-Ser422 Tau as well as ACL in HT22 cells (p-Tyr42 RhoA was reported to be active [p-Tyr42 Rho GTPase upon Aβ stimulation. P-Tyr42 Rho was increased by high concentration of Aβ (5–10 μM), while low concentration of Aβ (0.1–0.5 μM) reduced p-Tyr42 Rho levels , ACL, p-Ser9 GSK-3β (inactive), p-Thr180/Tyr182 p38 (active) and mSREBP1 were not influenced by Tat-C3  upon low concentrations of Aβ, but LiCl (GSK-3β inhibitor) did not impair p-Tyr416 Src. However, Tat-C3, Y27632 and LiCl did not effect on the levels of ACL, p-Ser422 Tau, and p-Tyr416 upon high concentration of Aβ (10 μM)  reduced ACL level, while high concentrations of Aβ (10 μM) increased ACL levels in PC12 cells A. Furthe22 cells B. In con22 cells B. As p-Te active , we trieo levels B. Herebyy Tat-C3 C. Consisu levels D. The re (10 μM) E. Inject (10 μM) F. Transc10 μM Aβ G.Fig. 4L3.5Aβ induced superoxide generation in time- and concentration-dependent manners in HT22 cells . Aβ alsop-Ser422 Tau was not altered by apocynin and DPI (possible inhibitors of NADPH oxidase) and NAC (ROS scavenger). However, high concentration of Aβ (10 μM)-maintained p-Ser455 ACL, ACL, p-Ser389 GSK-3β (inactive) and p-T180/Y182 p38 (inactive) were reduced by apocynin, DPI and NAC  and p21CIP1/WAF1 CDK inhibitors (CDKIs) were also markedly up-regulated by 10 μM Aβ (p-Ser1981 ataxia telangiectasia mutated (ATM) serine/threonine kinase was also increased, suggesting that DNA damage by superoxide activates ATM. Furthermore, si-ATM reduced p-Ser455 ACL, which was stimulated by high concentration of Aβ -mediated up-regulated  and NAC A. Transc and NAC B. Notabl and NAC C. In add and NAC D. Meanwh and NAC . In addi and NAC . In addi10 μM Aβ G. Markedon of Aβ , Fig. 6H3.6p-Tyr42 Rho in HT-22 cells (p-Tyr42 Rho (p-Tyr42 Rho induced by Aβ can be attributed to ROS. Injecting high concentrations of Aβ (100 μM) rather than 10 μM induced a greater degree of nuclear translocation of p-Tyr42 Rho in neurons of CA1 region of hippocampus in rat brain  (p-Tyr42 Rho antibody and NADK promoter primers demonstrated that p-Tyr42 Rho binds to the promoter of NADK upon Aβ stimulation (p-Tyr42 Rho exits in the nucleus upon Aβ treatment (10 μM) with western blotting. Remarkably, we demonstrated that Aβ (10 μM) induced an increase in the expression of NAD kinase (NADK)  restored NADK expression. However, reconstituted RhoA Y42F (dephospho-mimic) was not able to induce NADK expression, suggesting that p-Tyr42 residue of RhoA is crucial for NADK expression  D. ChIP Pmulation E. We vere (NADK) F, which e (NADK) G. Moreove (NADK) H and I. pression J.Fig. 6NSince not only NADPH oxidase but also FS requires NADPH as a hydrogen donor, we examined the level of superoxide production by inhibiting FS activity; cerulein, FS inhibitor enhanced superoxide production upon Aβ , suggest3.7p-Tyr42 Rho was also increased in CA1, CA2, DG (dentate gyrus), and EC  regions of human AD patients  were increased from total six specimens  and ACACB  were increased, while expression of SREBF2 , ACLY (ATP citrate lyase), FASN (fatty acid synthase), HMGCS1 (HMG-CoA synthetase), and HMGCR (HMG-CoA reductase) were significantly decreased in AD patients. ACACA  was not different from the control  in Aβ-mediated AD progression. Herein, we propose a new signalling pathway sequence as follows: low concentrations of Aβ → p-Tyr42 RhoA → ROCK → Src → p-Tyr216 GSK-3β → p-Tau. Although it was not clearly revealed how ROCK Ser/Thr kinase activates Src  is moved to the nucleus, where mSREBP1 plays a role as a transcriptional factor . Indeed,p-Tyr42 Rho by high concentration of Aβ. Therefore, the function of p-Tyr42 Rho under high concentrations of Aβ may be different from that under low concentrations of Aβ. It is notable that p-Tyr42 Rho is localized in the nucleus at high concentrations of Aβ  has been well known to be activated through the dissociation from Keap1 (Kelch ECH associating protein 1) upon ROS. Nrf2 translocates to nucleus, where it binds to antioxidant response element (ARE) and drives the expression of target genes such as heme oxygenase 1 (HMOX1/HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutamate-cysteine ligase (GCL) and glutathione-S-transferase (GST) [The ACL up-regulation is irrespective of an increase of ns of Aβ A, where ADK gene G, likelyse (GST) . Interesse (GST) ,49. In t5p-Ser422 Tau through activation of RhoA, Src, and Tyr216 GSK-3β (active form), high concentrations of Aβ caused cellular aging through superoxide, p-T180/Y182 p38, p-Ser389 GSK-3β (inactive form), mSREBP and ACL activation. In particular, p-Tyr42 RhoA is critical for both the elevation of p-Ser422 Tau and the expression of ACL and NADK. In particular, p-Tyr42 RhoA is localized in nucleus, where regulates expression of specific genes in response to Aβ. In this context, we propose a novel mechanism by which Aβ contributes to neuronal dysfunction through two different signalling pathways of p-Ser422 Tau and ACL-mediated neuronal aging depending on Aβ concentrations (We revealed that both high and low concentrations of Aβ are detrimental; while low concentrations of Aβ leads to trations G.None.


This conformation promotes the formation of a C(4) hydrogen-bonded chain propagating along the [010] direction.In the title compound, the amide functional group –C(=O)NH– adopts a  9H10ClNOS, the amide functional group –C(=O)NH– adopts a trans conformation with the four atoms nearly coplanar. This conformation promotes the formation of a C(4) hydrogen-bonded chain propagating along the [010] direction. The central part of the molecule, including the six-membered ring, the S and N atoms, is fairly planar (r.m.s. deviation of 0.014). The terminal methyl group and the C(=O)CH2 group are slightly deviating out-of-plane while the terminal Cl atom is almost in-plane. Hirshfeld surface analysis of the title compound suggests that the most significant contacts in the crystal are H⋯H, H⋯Cl/Cl⋯H, H⋯C/C⋯H, H⋯O/O⋯H and H⋯S/S⋯H. π–π interactions between inversion-related molecules also contribute to the crystal packing. DFT calculations have been performed to optimize the structure of the title compound using the CAM-B3LYP functional and the 6–311 G basis set. The theoretical absorption spectrum of the title compound was calculated using the TD–DFT method. The analysis of frontier orbitals revealed that the π–π* electronic transition was the major contributor to the absorption peak in the electronic spectrum.In the title compound, C Hirshfeld surface analysis was used to investigate the interactions within the crystal structure and DFT calculations were performed to study the frontier molecular orbitals of the title compound and also its electronic properties.Methylthioanilines are a class of S- and N- heterocyclic compounds that are widely used in antimicrobial applications , 0.324 (3) and 0.489 (4) Å for atoms C2, O1 and C1, respectively] while the terminal Cl atom is almost in-plane [−0.007 (3) Å] as a result of the N1—C2—C1—Cl1 torsion angle of −150.97 (18)°. The amide functional group adopts a trans conformation with the four atoms nearly coplanar as shown by the O1—C2—N1—H1 torsion angle of −176.5 (19)°. An intramolecular C—H⋯O contact is observed . A view along the a axis showing the unit-cell packing is shown in Fig. 2a while the hydrogen-bonded chain is illustrated in Fig. 2b. Apart from the hydrogen-bonding interactions, π-π stacking is observed between inversion-related molecules. The distance between the ring centroids is 3.8890 (14) Å while the distance between the mean planes is 3.3922 (10) Å (slippage 1.904 Å).The main feature of the crystal packing is the presence of an N—H⋯O hydrogen-bonded chain along the n Table 1 with graCrystal Explorer 17.5 , C⋯S/S⋯C (2.1%), H⋯N/N⋯H (2.1%) and C⋯C (2.8%).The intermolecular interactions in the crystal of the title compound were investigated by performing a Hirshfeld surface (HS) analysis  basis set in an ethanol solvent within the The DFT structure optimization of the compound was performed starting from the X-ray geometry at the CAM-B3LYP/6-311G level of theory in an ethanol solvent. The experimental and calculated geometrical parameters such as bond lengths and angles show good agreement although most of the calculated bond lengths are slightly longer than X-ray values (about 0.01 Å) because experimental values are for interacting molecules in the crystal lattice, whereas the computational method deals with an isolated molecule in the solvent phase.max) of the title compound was obtained using this method. As seen in Table 2max = 250 nm and the oscillator strength f = 0.7144 are due to the S0→S2 electronic transition with a wave function of two configurations [(HOMO→LUMO) and (HOMO→L+4)]. The transition from HOMO to LUMO is mainly responsible for the formation of the maximum wavelength at 250 nm  method to predict the absorption spectrum of the title compound in ethanol, also considering the excited states in the calculation. The maximum absorption wavelength et al., 2008aCc space group. Other structures with substituent(s) on the benzene ring include 2-chloro-N-acetamide (II) acetamide (III) acetamide (IV)  hydrogen-bond chain involving the primary amide functional group. This feature was also observed in the crystal structures of 2,2-chloro-N-phenylacetamide derivatives, viz. 2,2-dichloro-N-acetamide (V) acetamide (VI) acetamide] (VII) . The organic layer was dried with anhydrous Na2SO4. The mixture product was purified by column chromatography using 9:1 CH2Cl2/EtOAc as an eluent, affording a light-brown solid, yield 42%. Light-brown crystals were grown by evaporating a solution of the title compound in a mixture of dichloromethane and hexane (1:1) at room temperature. 1H NMR : δ 8.23 , 7.51 , 7.29 , 4.20 , 2.50 . Analysis calculated: C9H10ClNOS: C, 50.11; H, 4.67; N, 6.49 Found: C, 50.44; H, 4.69; N, 6.50The title compound was prepared by combining 4-(methylthio)aniline (5.0 g), chloroacetylchloride (4.30 mL) and triethylamine (7.50 mL) in dichloromethane (10 mL) at a controlled temperature using an ice bath. After stirring under an Nd(C—H) = 0.95 Å and Uiso(H) = 1.2Ueq(C) for aromatic and d(C—H) = 0.98 Å, Uiso(H) = 1.5Ueq(C) for methyl H atoms. The N-bound H atom (H1) was located in a difference-Fourier map and freely refined.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020002960/zq2250sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020002960/zq2250Isup3.hklStructure factors: contains datablock(s) I. DOI: 1987798CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, two sets of inversion-related C—H⋯π(ring) interactions form stacks of molecules extending along the a-axis direction.The 5-bromopentyl chain is oriented so that the bromine atom is  20H24Br2N4, the imidazopyridine moiety is not planar as indicated by the dihedral angle of 2.0 (2)° between the constituent rings; the 4-dimethylaminophenyl ring is inclined to the mean plane of the imidazole ring by 27.4 (1)°. In the crystal, two sets of C—H⋯π(ring) interactions form stacks of molecules extending parallel to the a-axis direction. Hirshfeld surface analysis indicates that the most important contributions to the crystal packing are from H⋯H (42.2%), H⋯C/C⋯H (23.1%) and H⋯Br/Br⋯H (22.3%) interactions. The optimized structure calculated using density functional theory (DFT) at the B3LYP/ 6–311 G level is compared with the experimentally determined structure in the solid state. The calculated HOMO–LUMO energy gap is 2.3591 eV.In the title molecule, C Besides the synthesis, we also report the molecular and crystal structures along with a Hirshfeld surface analysis and a density functional theory (DFT) computational calculation carried out at the B3LYP/6–311 G level.Given the wide range of theraputic applications for such compounds, we have already reported a route for the preparation of imidazo where Cg3 is the centroid of the C7–C12 phenyl ring et al., 2007a, while those delineated into H⋯H, H⋯C/C⋯H, H⋯Br/Br⋯H, H⋯N/N⋯H, C⋯Br/Br⋯C, N⋯Br/Br⋯N and N⋯C/C⋯N contacts are illustrated in Fig. 5b–h, respectively, together with their relative contributions to the Hirshfeld surface. The most important interaction is H⋯H, contributing 42.2% to the overall crystal packing, which is reflected in Fig. 5b as widely scattered points of high density due to the large hydrogen content of the molecule, with the tip at de = di = 1.18 Å. In the presence of C—H⋯π interactions, the pair of characteristic wings in the fingerprint plot delineated into H⋯C/C⋯H contacts (23.1% contribution to the HS), Fig. 5c, has the tips at de + di = 2.76 Å. The pair of scattered points of spikes in the fingerprint plot delineated into H⋯Br/Br⋯H, Fig. 5d (22.3%), have the tips at de + di = 2.95 Å. The H⋯N/N⋯H contacts, Fig. 5e (10.1%), have the tips at de + di = 2.56 Å. The C⋯Br/Br⋯C contacts, Fig. 5f, contribute 1.2% to the HS and appear as a pair of scattered points of spikes with the tips at de + di = 3.50 Å. The N⋯Br/Br⋯N contacts, Fig. 5g, contribute 1.1% to the HS appearing as pair of scattered points of spikes with the tips at de + di = 3.59 Å. Finally, the N⋯C/C⋯N contacts, Fig. 5h, make only 0.1% contribution to the HS and have a low-density distribution of points.In order to visualize the intermolecular interactions in the crystal of the title compound, a Hirshfeld surface (HS) analysis  are collated in Table 3H-imidazopyridine system. The energy band gap [ΔE = ELUMO − EHOMO] of the molecule is 2.3591 eV, and the frontier molecular orbital energies, EHOMO and ELUMO, are −3.1033 and −0.7442 eV, respectively.The optimized structure of (I)et al., 20162CH2X–  chain connecting a saturated nitrogen atom [corresponding to N2 in (I)] to an ortho position of the phenyl ring and so were considered less comparable to (I)R = Ph and R" = Br, examples are UCOXES methyl; Ouzidan et al., 2011aR′ = (2-oxooxazolidin-3-yl)ethyl; Ouzidan et al., 2011bR = 4-ClC6H4 and R" = Br there are two reports of the compound with R′ = 1-octyl-1H-1,2,3-triazol-4-yl)methyl [XITLUK A search of the Cambridge Structural Database -N,N-dimethylaniline , 2.2 equivalents of potassium carbonate  and 0.2 equivalents of tetra-n-butyl ammonium bromide (BTBA)  in 40 ml of DMF were added in small portions to 1.5 equivalent of the 1,6-dibromododecanedihalogenated reagent, and the mixture was stirred magnetically at room temperature for 48 h. After removal of the salts and evaporation of DMF under reduced pressure, the product was subjected to separation by chromatography on a column of silica gel using a mixture of hexane/dichloromethane = 1/4 (v/v) as the mobile phase. Brown single crystals suitable for X-ray diffraction were obtained by evaporation of a dichloromethane/hexane solution (1:4 v/v).To a solution of 4-(6-bromo-3Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989020008889/wm5570sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989020008889/wm5570Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020008889/wm5570Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020008889/wm5570Isup4.cmlSupporting information file. DOI: 2013416CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N-(4-methoxyphenyl)piperazine and its 3,5-dinitrobenzoate, picrate, and monohydrated 4-aminobenzoate salts are, respectively, one-, zero-, two- and three-dimensional.The hydrogen-bonded assemblies in  N-(4-methoxyphenyl)piperazine (MeOPP), (I), and for its 3,5-dinitrobenzoate, 2,4,6-trinitrophenolate (picrate) and 4-aminobenzoate salts, (II)–(IV), the last of which crystallizes as a monohydrate. In MeOPP, C11H16N2O, (I), the 4-methoxyphenyl group is nearly planar and it occupies an equatorial site on the piperazine ring: the molecules are linked into simple C(10) chains by N—H⋯O hydrogen bonds. In each of the salts, i.e., C11H17N2O+·C7H3N2O6−, (II), C11H17N2O+·C6H2N3O7−, (III), and C11H17N2O+·C7H6NO2−·H2O, (IV), the effectively planar 4-methoxyphenyl substituent again occupies an equatorial site on the piperazine ring. In (II), two of the nitro groups are disordered over two sets of atomic sites and the bond distances in the anion indicate considerable delocalization of the negative charge over the C atoms of the ring. The ions in (II) are linked by two N—H⋯O hydrogen bonds to form a cyclic, centrosymmetric four-ion aggregate; those in (III) are linked by a combination of N—H⋯O and C—H⋯π(arene) hydrogen bonds to form sheets; and the components of (IV) are linked by N—H⋯O, O—H⋯O and C—H⋯π(arene) hydrogen bonds to form a three-dimensional framework structure. Comparisons are made with the structures of some related compounds.Crystal structures are reported for  N-(4-Methoxyphenyl)piperazine (MeOPP) has fairly recently emerged as a new addition to the range of designer drugs aimed at recreational use, and considerable effort has consequently been invested in the development of rapid and reliable methods for the detection in human fluids not only of MeOPP itself but also of its primary metabolites N-(4-hydroxyphenyl)piperazine and 4-hydroxyaniline piperazine derivatives. Thus, we have recently reported the synthesis and structures of a range of salts derived from MeOPP piperazine (I)N-(4-methoxyphenyl)piperazine (MeOPP), and compounds (II)Compound (I)et al., 1987In the salt (III)ca 10° in each case, as is often observed when alkoxyarene systems are planar or nearly so , 0.033 (3), 0.040 (6) and 0.277 (7) Å in (I)–(IV), respectively. Associated with this near co-planarity, the two exocyclic O—C—C angles differ by 1 screw axis to form a C(10) The assembly in the 3,5-dinitrobenzoate salt (II)5) Fig. 6. There aet al., 1998ab Gregson et al., 20002 three-centre system containing an C(8)C(11) direction 0] Fig. 7. In the on Fig. 8. The comon Fig. 8.n, 0.5, 1) alternate with n + 0.5, 0.5, 1), where n represents an integer in each case e Fig. 10. In the e Fig. 10 link then Fig. 11.There are two C—H⋯π(arene) interactions in the structure of compound (IV)Hence the supramolecular aggregation is zero-, one-, two- and three-dimensional in compounds (II)et al., 2009et al., 2019versus the continuous aggregation in (VI) is not obvious. The electronic delocalization in the anion of (III)et al., 20196H4COO)−, where - = H, F, Cl or Br, compounds (VIII)–(XI), in all of which the 4-methoxyphenyl unit exhibits disorder piperazin-1-ium chloride (V) (Zia-ur-Rehman N-[4-Methoxyphenyl]piperazine (I)v/v).3) or 0.97 Å (CH2), and with Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups which were permitted to rotate but not to tilt, and 1.2 for all other H atoms bonded to C atoms. For the H atoms bonded to N atoms in (I)Uiso(H) = 1.2Ueq(N) giving the N—H distances shown in Table 1Uiso(H) = 1.2Ueq(N), while for those bonded to O in (IV)Uiso(H) = 1.5Ueq(O), giving the distances shown in Table 1ijU components. In the absence of significant resonant scattering, the correct orientation of the structure of (I)x parameter  − (I−)]/[(I+) + (I−)] was −0.5 (8), so that the correct orientation is indeterminate  global, I, II, III, IV. DOI: 10.1107/S2056989020002844/zl2772Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020002844/zl2772IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989020002844/zl2772IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989020002844/zl2772IVsup5.hklStructure factors: contains datablock(s) IV. DOI: Click here for additional data file.10.1107/S2056989020002844/zl2772Isup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020002844/zl2772IIsup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020002844/zl2772IIIsup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020002844/zl2772IVsup9.cmlSupporting information file. DOI: 1987252, 1987251, 1987250, 1987249CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The asymmetric unit of the title salt consists of an olanzapinium cation, an independent 2,5 dihydroxybenzoate anion and a solvent isopropyl alcohol molecule. The central seven-membered heterocycle is in a boat conformation, while the piperazine ring displays a distorted chair conformation. The dihedral angle between the benzene and thiene rings flanking the diazepine ring is 52.58 (19)°. In the crystal, the anions and cations are connected by N—H⋯O and O—H⋯O hydrogen bonds, forming a three-dimensional network. 17H21N4S+·C7H5O4−·C3H7OH, consists of an olanzapinium cation, an independent 2,5-dihydroxybenzoate anion and a solvent isopropyl alcohol molecule. The central seven-membered heterocycle is in a boat conformation, while the piperazine ring displays a distorted chair conformation. The dihedral angle between the benzene and thiene rings flanking the diazepine ring is 52.58 (19)°. In the crystal, the anions and cations are connected by N—H⋯O and O—H⋯O hydrogen bonds, forming a three-dimensional network.The asymmetric unit of the title salt, C Olanzapine is structurally similar to clozapine, but is classified as a thienobenzodiazepine. Reviews on olanzapine in the management of bipolar disorders  as well as associated cognitive deficits and are virtually devoid of extrapyramidal symptoms .The crystal structures of 2-methyl-4-(4-methylpiperazin-1-yl)-10Q = 0.715 (3) Å while the six-membered piperazine ring, N3/C13/C14/N4/C15/C16, adopts a distorted chair conformation with puckering parameters Q = 0.564 (3) Å, θ = 175.3 (3)°, φ = 200 (4)°. The dihedral angle between the benzene and thiene rings flanking the diazepine ring is 52.58 (19)°. This is similar to the values observed in the related structure olanzapinium dipicrate (II) [58.7 (9)°] . The dihedral angles between the plane of the four C atoms in the piperazine ring and the planes of the benzene and thiophene rings are 27.04 (13) and 33.36 (18)°, respectively. In the 2,5-dihydroxybenzoate, the mean plane of the C18–O1–O2 group is twisted by 4.7 (5)° from that of the benzene ring (C19–C24). The bond lengths and bond angles of the thiene and piperazine rings of compound (I)et al., 2013et al., 2005A perspective view of (I)et al., 2013et al., 2005The superimposed fit piperazin-1-ium 2,4,6-trihydroxybenzoate, and SUKPOG, 1-methyl-4-piperazin-1-ium 3,4,5-trihydroxybenzoate dihydrate; Sarmah et al., 2020H-thienobenzodiazepin-4-yl) piperazin-1-ium 4-hydroxybenzoate acetonitrile solvate, FABJIE, 1-methyl-4- piperazin-1-ium 2,5dihydroxybenzoate, FABJEA, 1-methyl-4-piperazin-1-ium 2,4-dihydroxybenzoate and FABJOK, 1-methyl-4- piperazin-1-ium 2,6dihydroxybenzoate; Sarmah et al., 2016H-thienobenzodiazepin-4-yl) hexahydropyrazin-1-ium nicotinate; Ravikumar et al., 2005et al., 2020et al., 2011aet al., 2020et al., 2016; FHIRYUE, HIRZAL, HIRZEP and HIRZIT; Thakuria et al., 2013et al., 2013et al., 2018et al., 2005). Among them, the crystal structures of PEWQEQ, PEWQAM, HIRZIT, FABJUQ, SUKPIA, SUKPOG, FABKEB, HIRZEP and PEWQAM contain solvent molecules.A search of the Cambridge Crystallographic Database  diagram, a tool to visualize π–π stacking interactions, for the cation, anion and solvent molecule is shown in Fig. 7de+ di = ∼2.3 Å). The second highest contribution is from H⋯C/C⋯H contacts (21.8%) and is indicated by the broad wing-like structure at de+ di = ∼2.6 Å. The symmetrical sharp spikes at de+ di = ∼1.6 Å are attributed to H⋯O/O⋯H contacts (14.3%).The HS analysis  and 2,5-dihydroxybenzoic acid  were dissolved in 20 mL of isopropyl alcohol and stirred magnetically for 5 h at 330 K. The mixture was kept aside for two days at room temperature and the salt formed was filtered off and dried. The compound was recrystallized from (1:1) isopropyl alcohol/DMF by slow evaporation at room temperature (m.p. 373–375 K).Uiso(H) = 1.5Ueq(C) for methyl H atoms and = 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698902000818X/mw2162sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698902000818X/mw2162Isup2.hklStructure factors: contains datablock(s) I. DOI: 2010899CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: