In the crystal structures of the two compounds, pronounced π–π stacking dominates the packing.2,3,8,9,14,15-Hexamethyl- and 2,3,8,9,14,15-hexaphenyl-5,6,11,12,17,18-hexazatrinaphthylene (HATNMe 6 and HATNPh6), are reported. Whereas the structure of the methyl-substituted derivative (HATNMe6) contains no solvent molecules (C30H24N6), the hexaphenyl-substituted structure (HATNPh6) contains two molecules of dichloromethane (C60H36N6·2CH2Cl2). This class of planar bridging ligands is known for its electron-deficient systems and its ability to form π–π stacking interactions. Indeed, in both crystal structures strong π–π stacking interactions are observed, but with different packing features. The dichloromethane molecules in the crystal structure of HATNPh6 are situated in the voids and are involved in C—H⋯N contacts to the nitrogen atoms of the pyrazine units.The crystal structures of two substituted HATN (hexaazatrinaphthylene) derivatives, namely 2,3,8,9,14,15-hexamethyl- and 2,3,8,9,14,15-hexaphenyl-5,6,11,12,17,18- hexazatrinaphthylene (HATNMe The central six-membered ring of 1 exhibits three longer  and three shorter  C—C bonds. The C—C bonds at the annulated benzene rings show differences in bond lengths. While the outermost bonds  are elongated (average 1.438 Å) the bonds to the left and right of these bonds  are shortened (average 1.366 Å).The title compound HATNMery Fig. 1. The mol6 (2) crystallizes with two molecules of CH2Cl2 in the triclinic space group P2, like in HATNMe6 (1), exhibits three longer  and three shorter  C—C bonds. These distances are slightly shorter in comparison with HATN 6  above as well as for HATN  are elongated (average 1.449 Å) and the bonds to the left and right of these bonds  are shortened (average 1.379 Å).HATNPhll Fig. 2. The mol6 (1) and HATNPh6 (2), these molecules stack in layers in their respective crystal structures. In the crystal packing of HATNMe6 (1), a herringbone-like arrangement of molecules is observed  form centrosymmetric dimers that are stacked perfectly parallel by van der Waals interactions but with a parallel displaced π-stacking. The plane-to-plane distance  within a dimer of 3.2518 (5) Å is shorter compared to the corresponding distance in 1. This distance, as well as the short centroid-to-centroid distance of 3.4018 (7) Å are both at the lower limit of ranges known for metal complexes with aromatic nitrogen-containing ligands , with an angle of 16.8° and a shift of approximately 1 Å. Comparing the plane-to-plane distances of the title compounds with related derivatives like HATN 6  have the shortest contact and the shortest displacement in π-stacking. Further interactions between the terminal phenyl rings and the pyrazines rings interconnect the dimers. The dichloromethane solvent molecules are located near the electron lone pairs of the N atoms in the voids of the packed molecules. They bridge two molecules of 2 and consolidate the crystal packing through weak C—H⋯N hydrogen-bonding interactions  and a saturated K2CO3 solution (100 ml) overnight in order to remove all traces of acetic acid. After filtration and washing with water, the solid was dried in a vacuum to give 2 as a yellow solid in 72% yield. Crystals suitable for single crystal X-ray diffraction were obtained by slow evaporation of a CH2Cl2 solution of 2.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018000725/wm5431sup1.cifCrystal structure: contains datablock(s) 1, 2. DOI: 10.1107/S2056989018000725/wm54311sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989018000725/wm54312sup3.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989018000725/wm54311sup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018000725/wm54312sup5.cmlSupporting information file. DOI: 1816408, 1816407CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Regulatory T cells (Tregs) play a pivotal role in maintaining immunological tolerance, but they can also play a detrimental role by preventing antitumor responses. Here, we characterized T helper (Th)-like Treg subsets to further delineate their biological function and tissue distribution, focusing on their possible contribution to disease states. RNA sequencing and functional assays revealed that Th2-like Tregs displayed higher viability and autocrine interleukin-2 (IL-2)-mediated activation than other subsets. Th2-like Tregs were preferentially found in tissues rather than circulation and exhibited the highest migratory capacity toward chemokines enriched at tumor sites. These cellular responses led us to hypothesize that this subset could play a role in maintaining a tumorigenic environment. Concurrently, Th2-like Tregs were enriched specifically in malignant tissues from patients with melanoma and colorectal cancer compared to healthy tissue. Overall, our results suggest that Th2-like Tregs may contribute to a tumorigenic environment due to their increased cell survival, higher migratory capacity, and selective T-effector suppressive ability.  •Memory Tregs can be classified as T helper-like Tregs •Human Th2-like Tregs exhibit the highest viability and IL-2-mediated activation•Th2-like Tregs are the subset with the highest chemotaxis toward CCL17/22•Th2-like Tregs are enriched at tumor sites in melanoma and colorectal cancer Halim et al. provide a comprehensive transcriptomic and functional analysis of circulating Th-like Tregs, revealing unique features in Th2-like Tregs and a significant enrichment of this subset in patients with melanoma and colorectal cancer. This suggests that Th2-like Tregs play a major role in maintaining a tumorigenic environment. In the peripheral circulation, the Treg population is composed of thymic-derived Tregs and Tregs that are induced in the periphery following T cell receptor (TCR) stimulation in a specific cytokine microenvironment (+ T helper (Th) cells. These new subpopulations, coined Th-like Tregs, express chemokine receptors CXCR3, CCR6, and CCR4, typically expressed by T-bet+-Th1, RORγt+-Th17, and GATA3+-Th2, respectively. The shared homing receptor distribution causes the appropriate co-localization of cell populations in peripheral tissue (Regulatory T cells (Tregs) are a subpopulation of T cells that elicit regulatory function by establishing and maintaining immunological tolerance and regulating immune homeostasis . In humaironment . Human Tironment . Furtherironment , as therironment . Thus, ul tissue . CCR4 mel tissue . CXCR3 ml tissue . The expl tissue . Underst+IL17+ T cells detected in colorectal cancer have the ability to suppress tumor-specific CD8+ T cells  were used to identify Th-like Treg lineages, as they contain functional representations of Th-like cells from all tissues . Total Tf CD45RA A. Using R3−CCR6− A. In conculation A. We theculation B and S1Bculation C. Of notculation D and S1Eculation C. Thus, culation D revealeculation . Gata-3,culation F and S2Aculation G. In linculation G. In addculation G. Higherculation G. The prculation B. Thus, FoxP3 has been shown to be a pro-apoptotic factor in developing Tregs in the absence of common gamma chain (γc)-dependent cytokine signals . Since Tint, and unlike Tregs, they exhibited a substantial percentage of CCR4− cells  after informed consent was approved. Patients with colorectal cancer  and melanoma  were consented in accordance with the Declaration of Helsinki. PBMCs were isolated by density-gradient centrifugation. Isolation protocols of mononuclear cells and research ethics for each tissue are described in +T Cell Enrichment Cocktail was used to isolate CD4+ T cells from leukapheresis cone blood  obtained from anonymous healthy donors. After negative isolation of CD4+ T cells, CD25 MicroBeads II were used to separate Tregs from Teffs. Tregs and Teffs were then sorted on a BD FACSAria I.RosetteSep Human CD4PBMCs and mononuclear cells obtained from tissues were stained with anti-CD4, CD25, CD127, CXCR3, CCR4, CCR6, CD45RA, CD3, CD8, and CCR8 for 30 min at 4°C in the dark. Transcription factor staining was then performed with the Foxp3/Transcription Factor Staining Buffer Set using anti-FoxP3, GATA3, T-bet, and RORγτ for 30 min at 4°C in the dark. Samples were acquired on LSR Fortessa and files analyzed using FlowJo (Tree Star). Gates were set based on biological controls and fluorescence minus one controls.5) were activated with CD3/CD28 beads (ratio 1:4) for 72 hr. Cells were lysed in TRIzol, and RNA was isolated with Direct-Zol RNA MicroPrep w/Zymo-Spin columns. RNA-seq was performed using the QIAGEN Human Inflammation and Immunity Transcriptome RNA targeted panel. Samples were quantified with the Agilent High Sensitivity DNA Kit and sequenced with the Illumina MiSeq using MiSeq Reagent Kit v3 (150-cycle) (Illumina). Principal-component analysis, volcano plots, and pathway analysis were performed using QIAseq targeted RNA data analysis tools (QIAGEN) and Partek Genomics Suite software, version 6.6.Fluorescence-activated cell sorting (FACS)-sorted Th-like Tregs (2 × 105), total memory Tregs (0.5 × 105) and Teffs (1 × 105) were stimulated with anti-CD3/CD28 beads at a 4:1 (cell/bead) ratio in the absence or presence of the neutralizing antibodies anti-IL-2 , anti-IL-4, anti-IFN-γ, or anti-IL-17  or in the presence of different concentrations of exogenous IL-2 (Novartis). After 16 hr, STAT5 and p53 were evaluated using western blot. After 72 hr, viability and apoptosis were evaluated using the LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit and Annexin V, and supernatants were used to detect human T cell cytokine production.FACS-sorted Th-like Tregs  ratio. Cells were activated with anti-CD3/CD28 beads at a 40:1 (cell/bead) ratio. Cellular proliferation was assessed after 4 days by flow cytometry, and files were analyzed using FlowJo. The data are presented as division index  obtained from FlowJo analysis. Supernatant was used to detect human T cell cytokine production using BD Cytometric Bead Array.FACS-sorted Th-like Teff subpopulations were labeled with 5 μM Cell Trace Violet for 37°C for 15 min. 1 × 10+ memory Teffs and unstained memory Tregs were sorted and rested prior experiment. After resting, 5 × 104 Teffs + 5 × 104 Tregs in 50 μl X-VIVO15 serum-free medium were placed in the top chamber. The bottom chambers were filled with 100 μl X-VIVO15 serum-free only; with CCL17 + CCL22, CCL20, or CXCL10; or with a combination of all of them . After 1 hr at 37°C, cells were harvested from bottom compartments, stained with anti-CXCR3, anti-CCR4 and anti-CCR6, counted using CountBright Absolute Counting Beads and analyzed by flow cytometry. The percentage of migration for each subset was calculated as (number of Th cells in the bottom chamber after 60 min × 100)/initial number of Th cells in the top chamber.T cell migration was assessed using a 5-μm-pore Transwell filter system. The top chambers were incubated with ICAM (1 μg/mL) overnight at 37°C. Cell Trace Violet∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.Statistical tests were performed using Prism 7 software (GraphPad). Data are expressed as mean ± SD or SEM where applicable using individual values, bar charts, or boxplots. A repeated-measures (RM) two-way ANOVA was used to compare two related variables between Th-like subsets. An RM one-way ANOVA was used to compare one related variable between Th-like Tregs. An ordinary one-way ANOVA was used to compare CCR8 expression between Th-like Tregs. A two-tailed t test was used to compare tumor specimens. Post hoc tests were used as indicated in the figure legends. p values are reported as follows: Conceptualization, E.N.-L., L.H., and G.L.; Methodology, E.N.-L. and M.R.; Formal Analysis, E.N.-L. and R.M.; Investigation, E.N.-L., L.H., M.R., R.M., I.C., P.P., N.G., S.-J.H., M.Y., M.H., and O.H.; Resources, G.L., R.F.H., W.J., B.H., G.E.D., S.N.K., and N.P.; Writing – Original Draft, E.N.-L. and L.H.; Writing – Review & Editing, E.N.-L., L.H., M.R., R.M., I.C., P.P., N.P., R.I.L., and G.L.; Visualization, Supervision and Project Administration, E.N.-L.; Funding Acquisition, G.L.


These have symmetry-equivalent magnesium atoms bridged by μ-iodide ligands with very similar Mg—I distances.The crystal structure of a dimeric β-diketiminate magnesium(II) complex crystallizes as two independent molecules, each with 2/ 2N,N′)magnesium(II)] toluene sesquisolvate, [Mg2(C29H41N2)2I2]·1.5C7H8, crystallizes as two independent molecules, each with 2/m crystallographic site symmetry, located at Wyckoff sites 2c and 2d. These have symmetry-equivalent magnesium atoms bridged by μ-iodide ligands with very similar Mg—I distances. The two Mg atoms are located slightly below (∼0.5 Å) the least-squares plane defined by N–C—C–N atoms in the ligand scaffold, and are approximately tetrahedrally coordinated. One and one-half toluene solvent molecules are disordered with respect to mirror-site symmetry at Wyckoff sites 4i and 2a, respectively. In the former case, two toluene molecules interact in an off-center parallel stacking arrangement; the shortest C to C′ (π–π) distance of 3.72 (1) Å was measured for this interaction.The solid-state structure of a dimeric β-diketiminate magnesium(II) complex is discussed. The compound, di-μ-iodido-bis[phenyl]pent-3-en-2-ylidene}azanido-κ This nitrogen analogue of acac allows for modular electronic and steric tuning of the ligand framework by altering groups on the N atoms, which engenders marked stability in low-oxidation-state metals as described in the literature, notably by the late Professor Lappert  Å and 0.473 (3) Å out of the mean least-square plane defined by (N1–C1–C1–N1) or (N2–C21–C21–N2), which suggests predominantly κ2 bonding. Chelate-ring atoms C2 and C22 deviate from these planes by 0.127 (4) and 0.111 (4) Å, respectively. The coordination geometry is best described as stated; however, there are reports of low-lying p orbitals contributing to more pronounced deviations of the metal from the calculated plane of the ligand , MgI2 , and a magnetic stir bar. Diethyl ether  was condensed into the flask at 195 K by vacuum transfer, providing a cloudy beige solution. The reaction mixture was warmed to 273 K and stirred for three h. The flask was then warmed to room temperature and all volatiles were removed under reduced pressure. The resulting white residue was reconstituted in toluene and passed through a fine porosity frit, affording a clear-yellow filtrate. The filtrate was concentrated under vacuum and single crystals suitable for diffraction were grown from this concentrated toluene solution at 238 K. 1H NMR (CDCl3): δ 7.12 , 6.97 , 4.76 CH), 3.02 2), 1.57 CH), 1.04 , 0.78 . 13C{1H} NMR (CDCl3): δ 169.9 (NC(CH3)CH), 143.6 (aromatic C), 142.6 (aromatic C), 125.6 (aromatic CH), 123.8 (aromatic CH), 94.8 (NC(CH3)CH), 28.0 (CH(CH3)2), 25.6 (CH(CH3)(CH3)), 24.7 (CH(CH3)(CH3)), 24.4 (NC(CH3)CH). Analysis calculated for C58H82I2Mg2N4: C, 61.23; H, 7.26; N, 4.92. Found: C, 61.06; H, 6.98; N, 5.14.Under a dry, argon atmosphere, an oven-dried Schlenk flask was charged with lithium a and 4i and are disordered w.r.t. the 2/m and m symmetry, respectively; surprisingly both lie with their molecular planes perpendicular to the crystallographic mirror. Both toluene molecules are (at least approximately) coplanar with the typical orientations where the methyl carbons C47 and C57 lie close to C44 and C54 of the rings, consistent with interactions of aromatic solvents  I. DOI: 10.1107/S2056989016017394/zl2683Isup2.hklStructure factors: contains datablock(s) I. DOI: 1512965CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Mechanistically, we found that low expression of the antioxidant glutathione in CD27− γδ17 T cells renders them particularly susceptible to neutrophil-derived reactive oxygen species (ROS). Consistently, superoxide deficiency, or the administration of a glutathione precursor, rescued CD27− Vγ6+ γδ17 T-cell proliferation in vivo. Moreover, human Vδ1+ γδ T cells, which contain most γδ17 T cells found in cancer patients, also displayed low glutathione levels and were potently inhibited by ROS. This work thus identifies an unanticipated, immunosuppressive yet antitumoral, neutrophil/ROS/γδ17 T-cell axis in the tumor microenvironment.Interleukin 17 (IL-17)–producing γδ T cells (γδ17 T cells) have been recently found to promote tumor growth and metastasis formation. How such γδ17 T-cell responses may be regulated in the tumor microenvironment remains, however, largely unknown. Here, we report that tumor-associated neutrophils can display an overt antitumor role by strongly suppressing γδ17 T cells. Tumor-associated neutrophils inhibited the proliferation of murine CD27 + γδ T cells express very low levels of the antioxidant, glutathione, and are very sensitive to reactive oxygen species (ROS), thus revealing their Achilles’ heel. Indeed, as ROS-producing neutrophils accumulate within tumors, they inhibit IL-17+ γδ T-cell proliferation and thereby suppress their pro-tumoral activities. We extended these findings, obtained in mouse models of cancer, to human γδ T cells and therefore believe that the modulation of local levels of oxidative stress may have important therapeutic implications.Tumors are infiltrated by many immune cells that influence many aspects of cancer progression and outcome, including tumor growth, invasion of healthy surrounding tissues, formation of metastasis, and response to treatments. Among tumor-infiltrating lymphocytes, γδ T cells play dual functions in the tumor milieu; whereas those that produce the antitumor cytokine interferon-γ are protective, their counterparts that make interleukin 17 (IL-17) support tumor growth. It is therefore critical to understand which mechanisms may limit IL-17–biased γδ T-cell responses. In this study, we unexpectedly found that IL-17 Amongst such immune populations, γδ T cells are known to contribute to protective responses because of their potent ability to kill tumor cells and to produce cytokines like interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) –5, whichn cancer  and squan cancer  patientsn cancer .+ T-cell responses. Thus, γδ17 T cells have been extensively shown to interact with myeloid cells that counteract tumor immune surveillance, and instead promote cancer progression.The pro-tumoral function of γδ17 T cells was shown to result from either direct support of tumor cell survival, through the interleukin 6 (IL-6)–signal transducer and activator of transcription 3 (STAT3) axis ,15. MoreThis notwithstanding, a large-scale analysis of thousands of tumor samples from 39 cancer types indicated that γδ T cells are globally associated with a good prognosis , which m− γδ T-cell compartments [+ or Vγ6+ T-cell receptors (TCRs) [+ γδ17 T cells typically populate secondary lymphoid organs , their Vγ6+ counterparts leave the fetal thymus to become tissue-resident, long-lived, and self-renewing cells that respond in situ [+ γδ17 T cells are abundant, such as the dermis, tongue, lung, liver, uterus, and peritoneal cavity [− Vγ6+ γδ17 T cells proliferated extensively in the peritoneal cavity following the transplantation of ID8 ovarian cancer cells, thereby constituting the major source of pro-inflammatory and pro-angiogenic IL-17 that promoted tumor cell growth [In mice, γδ17 T cells are comprised in discrete thymic and peripheral CD27artments  and can s (TCRs) . These ss (TCRs) ,23 and ds (TCRs) . Namely, in situ –26. Thisl cavity . Interesl growth .Following that study, we have investigated the contribution of γδ17 T cells to different tumor types developing in the same environment. Unexpectedly, we found that pro-tumoral γδ17 T cells failed to respond to discrete tumor challenges due to neutrophil-mediated suppression, which therefore limited tumor growth. We went on to dissect the molecular mechanisms underlying this unanticipated neutrophil/γδ17 T-cell cross talk in experimental mouse models of cancer and found an exquisite sensitivity of γδ17 T cells to reactive oxygen species (ROS)-induced oxidative stress in the tumor microenvironment.+ and CD4+ T cells accumulated significantly (+Ly6G+Ly6Cint) and monocytes (CD11b+Ly6G-Ly6C+) accumulated, respectively, 40- and 20-fold within the leukocyte infiltrate (CD45+ cells)  . Althoug+ cells) , we none+ cells) . Criticaing mice . Given tl cavity , whereasing mice , thus su+ cells) compared to the tumor-free liver tissue , lacks mature neutrophils (in the periphery and in the bone marrow) without impacting on lymphopoiesis nor on T- and B-Cell functions [Genista mice displayed low frequencies of neutrophils, and the few remaining tumor-associated neutrophils expressed lower levels of the maturation markers, Ly6G and CD11b, when compared to littermate heterozygous controls (+ γδ T cells in the intrahepatic Hepa 1–6 model (Genista mice compared to their littermate controls (Il17−/− mice compared to neutrophil-depleted wild-type (WT) mice  in which tumor growth is increased in the presence of IL-17 . We implr tissue . We nextunctions ,28. Conscontrols . Importa–6 model . Moreove+ leukocytes) was increased upon neutrophil depletion/deficiency in both tumor models, while the frequency of IFN-γ–producing cells remained unchanged −/− mouse strain that lacks the enzyme NADPH oxidase 2 (NOX2), which catalyzes the conversion of molecular oxygen to superoxide. We purified neutrophils from the peritoneal cavity of tumor-bearing Cybb−/− or WT mice and co-cultured them with anti-CD3/CD28-stimulated CD27− γδ T cells. Whereas WT neutrophils drastically inhibited the proliferation of CD27− γδ T cells, the latter were able to divide in the presence of Cybb−/− neutrophils, albeit not as efficiently as in the complete absence of neutrophils  mice, as we found that Vγ6+ and IL-17+ γδ T cells accumulated to significantly higher levels in Cybb−/− than in control mice , β subunit , as a potential gain-of-function approach. Indeed, NAC treatment was sufficient to lead to an accumulation of Vγ6ing mice . Taken t+ CD27− γδ T cells were especially affected by neutrophil-derived ROS, we assessed the effect of increasing concentrations of hydrogen peroxide (H2O2) and superoxide (O2.-) (generated by the xanthine/xanthine oxidase system) on the proliferation of CD27− and CD27+ γδ T-cell subsets in vitro. Both hydrogen peroxide and superoxide inhibited γδ T-cell proliferation, but CD27− cells were clearly more susceptible than their CD27+ counterparts (− γδ17 T cells might have lower capacity to detoxify ROS than CD27+ γδ T cells (or other T-cell subsets). Moreover, as γδ17 T cells expanded upon in vivo administration of NAC , as well as Gss, the gene that encodes for glutathione synthetase, were expressed less in IL-17+ T cells, which may explain the low glutathione pool in CD27− γδ T cells. Most other antioxidants, such as thioredoxins and peroxiredoxines, were also lower in IL-17+ γδ T cells. Altogether, this supports that differences in redox metabolism make γδ17 T cells more sensitive to oxidative stress than γδIFN-γ T cells.To understand why Vγ6terparts . These rn of NAC , and thi T cells . This maferation  compared T cells , CD8+ or T cells  in neutr T cells  were sel T cells . For exa+ γδ T cells, the main γδ T-cell subset associated with IL-17 production in human tumors [2O2, in contrast with their Vδ2+ γδ, CD8+, and CD4+ T-cell counterparts  or integrin beta chain-2 , which are characterized by low neutrophil counts in tissues, show increased tissue-resident γδ17 T cells [γδ17 T cells are known to enhance neutrophil mobilization in the context of several infections and also in response to tumors ,21,35. M T cells ,41.+ T-cell functions via secretion of IL-10 [+ γδ17 T cells and neutrophils that led to inducible nitric oxide synthase (iNOS)-dependent suppression of cytotoxic CD8+ T cells and promoted lung metastases [A dual role for neutrophils in cancer has been suggested –46, and of IL-10 , arginasof IL-10 , or reacof IL-10 . In facttastases .+ T cells, through NOX2-dependent ROS production. These discordant actions of neutrophils in different tumor models may rely on their relative ROS levels and differential impact on T-cell subsets. As suggested by our data, γδ17 T cells expressing low intracellular glutathione are particularly susceptible to oxidative suppression, whereas CD8+ T cells likely require greater ROS concentrations. Interestingly, Treg cells were also recently shown to be highly sensitive to oxidative stress in the tumor microenvironment, due in this case to a weak nuclear factor (erythroid-derived 2)-like 2 (NRF2)-associated antioxidant system [By contrast, in the peritoneal B16 and intrahepatic Hepa 1–6 tumor models, neutrophils inhibited γδ17 T cells, but not CD8t system , which mGenista mice, which lack mature neutrophils, indicate that it is the mature neutrophils that suppress γδ17 T cells. Thus, we propose that neutrophils can be suppressive and yet antitumoral by targeting γδ17 T cells, which is in line with their protective role, linked to IL-17 inhibition, in the murine Lewis lung carcinoma model [On the other hand, the pleiotropic roles of neutrophils may be associated with heterogeneous maturation and activation phenotypes in different tumor models as well as mouse backgrounds (such as FVB versus C57BL/6) . For exama model .+ T cells can be important IL-17 producers that favor cancer progression through induction of inflammation [+ T cells express low basal levels of glutathione and are highly susceptible to ROS. In line with this, human neutrophils from healthy donors have also been shown to impact circulating γδ T-cell activation and cytokine production and proliferation through production of ROS [In humans, Vδ1ammation  and recrammation . Consistn of ROS .2−), hydrogen peroxide (H2O2), hypochlorous acid (HCl), and hydroxyl radical, among others. Superoxide and hydrogen peroxide are the most common ROS involved in biological processes. Superoxide is rapidly dismutated to hydrogen peroxide or immediately reacts with surrounding molecules; hydrogen peroxide is more stable and can diffuse in the microenvironment and across cell membranes [ROS are short-lived molecules that originate from molecular oxygen and include superoxide  between Instituto de Medicina Molecular (iMM) and Instituto Português do Sangue e da Transplantação and were approved by the local ethical committee . All mouse experiments performed in this study were evaluated and approved by our institutional ethical committee (iMM-Orbea) and the national competent authority (DGAV) under the license number 019069. Briefly, euthanasia was performed by CO−/− and B6.TCRδ−/− mice were purchased from Charles River Laboratories. B6.Il17−/− mice were kindly provided by Fiona Powrie , with permission from Yoichiro Iwakura . Genista mice were imported from the Center of Immunology Marseille Luminy (France) and bred in house. Genista homozygous mice were used as a neutropenic model and were compared to their heterozygous littermate controls. Mice were maintained in specific pathogen-free facilities of iMM. Cybb−/− male mice and their respective C57Bl/6J controls were purchased from Jackson laboratories and maintained in specific pathogen-free facilities at the Francis Crick Institute. IFN-γ/IL-17 double-reporter mice, generated by crossing IFN-γ-YFP mice [C57Bl/6J (B6) WT mice and B6.TCRαYFP mice  with Il1YFP mice , were usYFP mice .6 Hepa 1–6 cells implanted intrahepatically in 20 μL PBS through surgical procedure. Mice were euthanized 2–3 weeks later, and tumors were extracted for subsequent analysis. We injected 5 × 104 B16-F0 tumor cells intraperitoneally in 100 μL of PBS. Tumor growth was evaluated in situ by bioluminescence imaging as previously described [6 ID8 cells were injected intraperitoneally. ID8 tumors were let to grow for 2 weeks, after which 5 × 104 B16 cells were inoculated i.p. One group of mice was injected with anti-Gr-1 as described below. Two weeks after B16 tumor inoculation (and 4 weeks upon ID8 injection), mice were euthanized and peritoneal exudate cells analyzed by FACS.For orthotopic hepatocellular carcinoma model, anesthetized mice received 1 × 10escribed . For proFor in vivo antibody depletion, 70 μg anti-Gr1 , 250 μg (Hepa 1–6 intrahepatic model) , 250 μg anti-Ly6G antibody , 1 mg anti-CD25 , 300 μg anti-CD115 , 70 μg isotype control , or PBS was injected i.p. at days 4, 8, and 12 post–tumor inoculation. For monocyte/macrophage depletion, 100 μL of clodronate liposomes (Liposoma B.V.) were injected s.c. or i.v. at days 4, 8, and 12 post–tumor inoculation.NAC (Sigma/Merck) was resuspended in PBS (pH = 7) and administrated i.p. every other day, from day 4 post–tumor injection, at a concentration of 15 mg/kg.Fingolimod  was given in the drinking water (2.5 μg/mL) from day 4 post–tumor inoculation.Blood leukocytes (buffy coat cells) were isolated by gradient centrifugation in Histopaque and each lymphocyte population was FACS-sorted in FACS Aria (BD Biosciences).Hepa 1–6 tumors were harvested, finely chopped, and digested with 1 mg/mL collagenase Type I, 0.4 mg/mL collagenase Type IV (Worthington), and 10 μg/mL DNase I (Sigma/Merck) for 30 minutes at 37 °C. Cell suspension was then filtered through a 100 μm nylon cell strainer . Peritoneal exudate cells were obtained from the lavage of the peritoneal cavity with 5 mL ice-cold DMEM with 10% (vol/vol) FCS. Erythrocytes were osmotically lysed using RBC Lysis Buffer (Biolegend). For surface staining, cells were Fc blocked with anti-CD16/32  and incubated for 45 minutes with antibodies and LIVE/DEAD Fixable Near-IR (Thermo Fisher Scientific) in complete RPMI medium. The following monoclonal antibodies were purchased from eBioscience/Thermo Fisher Scientific: anti-CD3ε , anti-CD4 (RM4-5), anti-CD11b (M1/70), anti-F4/80 (BM8), anti-MHC II (M5/114.15.2), anti-CD27 (LG.7F9), and anti-TCRγ4 (UC3-10A6); from Biolegend: anti-CD8α (53–6.7), anti-CD45 (30-F11), anti-TCRδ (GL3), anti-Ly6C (HK1.4), anti-Ly6G (1A8), anti-NK1.1 (PK136), and anti-TCRγ1 (2.11); and from BD Pharmigen: anti-CD44 (IM7).For T-cell intracellular cytokine staining, cells from tumor, PEC, or spleen were stimulated with 50 ηg/mL phorbol 12-myristate 13-acetate  and 1 μg/mL ionomycin (Sigma/Merck) for 3 hours at 37 °C in the presence of 10 μg/mL brefeldin-A (Sigma/Merck) and 2 μM monensin (eBioscience/Thermo Fisher Scientific). Cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer set (eBioscience/Thermo Fisher Scientific), following the manufacturer’s instructions, and then incubated for 30 minutes at room temperature, with the following antibodies from eBioscience/Thermo Fisher Scientific: anti-IFN-γ (XMG1.2), anti-IL-17 (TC11-18H10.1), Foxp3 (FJK-16s), and Ki67 (16A8). For BrdU staining, FITC BrdU Flow Kit (BD Pharmingen) was used following manufacturer’s instructions. For TCRγ6 (Vγ6) detection, staining with GL3 and 17D1 monoclonal antibodies  was performed as previously described . For AnnCells were acquired on a FACS Fortessa (BD Biosciences) or LSR II, sorted on FACS Aria, and data analyzed using FACS Diva or FlowJo software (Tree Star).−/− mice. Cell suspensions were stained with anti-CD3ε (145-2C11), anti-TCRδ (GL3), and anti-CD27 (LG.7F9) for 30 minutes at room temperature. CD27+, CD27− γδ T cells, CD4, and CD8 T cells were FACS-sorted and stained with 1 mM of Cell Trace Violet (Thermo Fisher Scientific) in PBS for 20 minutes at room temperature. Cells were incubated on plate-bound anti-CD3ε (145.2C11) (10 μg/mL) plus anti-CD28 mAb (37.51) (5 μg/mL) in the presence of IL-7 (50 ηg/mL) for 72 hours. IL-7 was from Peprotech and the antibodies were from eBiosciences/Thermo Fisher Scientific or BioLegend.Lymphoid (spleen and lymph nodes) were harvested from C57Bl/6J or B6.TCRαCybb−/− mice or from the bone marrow of tumor-free or tumor-bearing WT mice. For neutrophil purification, cells were stained with αGr-1–PE (RB6-8C5) at a concentration of 40 ηg/mL, and mouse anti-PE selection kit (STEMCELL Technologies) was used.Neutrophils were isolated from the peritoneal exudates of B16 tumor–bearing WT or 2O2, the superoxide-generating system xanthine/xanthine oxidase (Sigma/Merck), or catalase (Sigma/Merck).Alternatively, cells were cultured with different concentrations of H2O2. CTV dilution was assessed by FACS.Human cells were cultured with soluble anti-CD3  and IL-2 (10 ηg/mL) for 6 days in the presence of 100–300 μM of Hhprt or β2microglobulin was carried out using SYBR on ABI ViiA7 cycler (Applied Biosystems). The CT for the target gene was subtracted from the CT for endogenous references, and the relative amount was calculated as 2−ΔCT. Primer sequences were the following: nfe2l2 forward, GCAGCCATGACTGATTTAAGC, nfe2l2 reverse, CAGCCAGCTGCTTGTTTTC, gclc forward, GGCTCTCTGCACCATCACTT, gclc reverse, GTTAGAGTACCGAAGCGGGG, gclm forward, AGGAGCTTCGGGACTGTATCC, gclm reverse, GGGACATGGTGCATTCCAAAA, gpx1 forward, CAATGTAAAATTGGGCTCGAA, gpx1 reverse, GTTTCCCGTGCAATCAGTTC, gpx4 forward, TAAGAACGGCTGCGTGGT, gpx4 reverse, GTAGGGGCACACACTTGTAGG, gsr forward, ATCGTGCATGAATTCCGAGT, gsr reverse, GGTGGTGGAGAGTCACAAGC, gss forward, CACTATCTCTGCCAGCTTTGG, gss reverse, TTATTCAGGACATTGAGAACGTG, mdh2 forward, TGACCTGTTCAACACCAACG, mdh2 reverse, GATGGGGATGGTGGAGTTC, pgd forward, ATGGCCCAAGCTGACATTG, pgd reverse, GCACAGACCACAAATCCATGAT, prdx1 forward, GTTGGCCGCTCTGTGGATGAGAT, prdx1 reverse, ATCACTGCCAGGTTTCCAGCCAGC, prdx2 forward, GTTCTCCGGCCTAGGGCTCTCTC, prdx2 reverse, GCCGGAGGCCATGACTGCGTG, prdx3 forward, GAACCTGTTTGACAGACATACTGTG, prdx3 reverse, GGGGTGTGGAAAGAGGAACT, prdx4 forward, CTCAAACTGACTGACTATCGTGG, prdx4 reverse, CGATCCCCAAAAGCGATGATTTC, sod1 forward, TACTGATGGACGTGGAACCC, sod1 reverse, GAACCATCCACTTCGAGCA, srxn1 forward, AGTAGTAGTCGCCACCCTGG, srxn1 reverse, AGAGCCTGGTGGACACGAT, txn1 forward, TGCTACGTGGTGTGGACCTTGC, txn1 reverse, TCTGCAGCAACATCCTGGCAGT, txn2 forward, CGACCTTTAACGTCCAGGATG, txn2 reverse, ACTGTGCATGAAAGTCCACAAC, txnrd1 forward, ATGGACAGTCCCATCCCGGGA, txnrd1 reverse, GCCCACGACACGTTCATCGTCT, txnrd3 forward, CCAAGAAATATGGCTGGGAGT, txnrd3 reverse, TGTAGCCCCAGTTCAAGGAG.mRNA was prepared from FACS-sorted cell populations using High Pure RNA Isolation kit (Roche). Reverse transcription was performed with random oligonucleotides (Invitrogen) using Moloney murine leukemia virus reverse transcriptase (Promega) for 1 hour at 42 °C. Relative quantification of specific cDNA species to endogenous reference 2O2 was measured using OxiSelect Hydrogen Peroxide/Peroxidase Assay Kit (Cell Biolabs), following manufacturer’s instructions. Protein oxidation was measured by flow cytometry using the FlowCellect oxidative stress kit (Sigma/Merck), following manufacturer’s instructions. For glutathione quantification, cells were FACS-sorted from spleen and LN of C57Bl/6J or B6.TCRα−/− tumor-free mice and lysed in 5% metaphosphoric acid at a concentration of 2 × 106 cells per mL . Glutathione (GSSG/GSH) detection kit (Enzo Life Sciences) was used to quantify total glutathione according to manufacturer’s instructions.Ht test with 95% confidence intervals for unrelated samples. For paired samples, Wilcoxon-matched-pairs test was used. When more than two groups were compared, two-way ANOVA followed by Tukey HSD post hoc test was performed. Unless otherwise indicated, individual values and mean are plotted, or mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.No statistical methods were used to predetermine sample size. Statistics were done using nonparametric two-tailed Mann-Whitney test or, if both groups followed a normal distribution , using two-tailed unpaired Student S1 Data(XLSX)Click here for additional data file.S1 Fig(A) Neutrophil frequencies in peritoneal exudates of B16 tumor–bearing or ID8 tumor–bearing mice, assessed at week 2 (B16) or at weeks 2 and 8 (ID8) after tumor implantation. (B) IL-17+ γδ, CD27+ γδ T-cell frequency in peritoneal exudates of tumor-free, ID8 + B16 tumor–bearing and ID8 + B16 tumor–bearing mice depleted for neutrophils. Statistical analysis was performed by Mann-Whitney test.(TIF)Click here for additional data file.S2 Fig(A) Neutrophil, monocyte, and Treg cell frequencies in peritoneal exudates of B16 tumor–bearing mice treated with αGR-1, αLy6G, αCD115 + clodronate–containing liposomes and αCD25 mAbs. (B) Representative FACS plots of neutrophils and summary of Ly6G and CD11b MFI in neutrophils from Genista heterozygous or homozygous Hepa 1–6 tumor–bearing mice. mAb, monoclonal antibody; MFI, mean fluorescence intensity; Treg, regulatory T.(TIF)Click here for additional data file.S3 Fig(A) Representative FACS plots and frequency of IL-17+ cells and IFN-γ+ cells in the peritoneal exudates of B16 tumor–bearing (top) and Hepa 1–6 tumor–bearing mice (bottom), either in the presence (Neu +) or absence (Neu −) of neutrophils. Red and blue circles represent αGr-1 mAb-treated or PBS-treated C57BL/6 mice, respectively, whereas red and blue triangles represent Genista homozygous or littermate controls, respectively. Data were pooled from three independent experiments. (A) Representative FACS plots and summary chart of γδ T-cell and CD4+ T-cell contributions to the IL-17+ CD3+ pool, as well as their MFI in the absence of neutrophils (as in A) or in intraperitoneal B16 (top) or intrahepatic Hepa 1–6 (bottom) tumor models. Data were pooled from two independent experiments. Dotted lines link subsets from the same mouse. Statistical analysis was performed using Mann-Whitney test or Wilcoxon-matched-pairs signed rank test .(TIF)Click here for additional data file.S4 Fig(A) Apoptotic Vγ6+ T cells, assessed by annexin V and caspase 3/7 cleavage, in the peritoneal exudates of PBS or aGr-1 mAb-treated B16 tumor–bearing mice at days 9 and 13 post–tumor inoculation. Data were pooled from two independent experiments. (B) Frequency of Vγ6+, CD8+, and CD4+ T cells in the peritoneal exudates of PBS or αGr-1 mAb-treated or FTY720-treated PBS or αGr-1 mAb-treated B16 tumor–bearing mice. Statistical analysis was performed using two-way ANOVA followed by Tukey HSD post hoc test.(TIF)Click here for additional data file.S5 Fig(A) In vitro inhibition of CD27− γδ, CD27+ γδ, CD4, and CD8 T-cell proliferation in the presence of neutrophils from the peritoneal cavity of B16 tumor–bearing mice. (B) CD27− γδ T-cell proliferation cultured alone, in the presence of neutrophils from the peritoneal cavity of B16 tumor–bearing mice, with or without catalase.(TIF)Click here for additional data file.


II atom.The complex title molecule lies across an inversion centre and exhibits a slightly distorted octahedral coordination environment for the Co 7H7N4O2)2(H2O)4], comprises mononuclear molecules consisting of a CoII ion, two deprotonated theophylline ligands  and four coordinating water molecules. The CoII atom lies on an inversion centre and has a slightly distorted octahedral coordination environment, with two N atoms of two trans-oriented theophylline ligands and the O atoms of four water molecules. An intramolecular hydrogen bond stabilizes this conformation. A three-dimensional supramolecular network structure is formed by intermolecular O—H⋯O and O—H⋯N hydrogen bonds.The title complex, [Co(C H-purine-2,6-dione) belongs to the family of xanthines, which are purine derivatives. It is related to dietary xanthines caffeine and theobromine and is an important pharmacologic compound 2(H2O)4].In this study, we reacted theophylline with the CoII atom being coordinated in a slightly distorted octahedral environment by four aqua ligands in the equatorial sites and the imidazole ring N atoms of two 1,3-dimethyl-2,6-dioxo-7H-purin-7-ide ligands [N1 and N1i; see Table 1S(7) graph-set motif  Å and O14⋯O11iii = 2.773 (2) Å; see Table 2iv = 2.799 (2) Å; see Table 2In the crystal, the mononuclear units are connected into a layered arrangement parallel to (010). The coordinating water molecules are involved in various hydrogen-bonding interactions Table 2, includi2·6H2O  in water (10 ml) was then added. Pink single crystals of the title compound suitable for X-ray analysis were obtained after several months by slow evaporation of the solvent at room temperature.Theophylline  was dissolved in water (20 ml). An aqueous solution (10 ml) of NaOH  was added slowly. CoClUiso(H) values (in the range 1.2–1.5 times Ueq of the parent atom), after which the positions were refined with riding constraints  global, New_Global_Publ_Block, I. DOI: 10.1107/S2056989017011379/wm5408Isup2.hklStructure factors: contains datablock(s) I. DOI: 1566195CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Enterococcus faecium HDRsEf1 (Ef1) has been shown to have positive effects on piglet diarrhoea, but the mechanism has not yet been elucidated. In this study, using the IPEC-J2 cell line to mimic intestinal epithelial cells and enterotoxigenic Escherichia coli (ETEC) K88ac as a representative intestinal pathogen, the mechanism underlying Ef1 protection against an enteropathogen was investigated. The results demonstrated that Ef1 was effective in displacing K88ac from the IPEC-J2 cell layer. Moreover, Ef1 and its cell-free supernatant (S-Ef1) modulate IL-8 released by IPEC-J2 cells. Ef1 and its cell-free supernatant showed the potential to protect enterocytes from an acute inflammatory response. In addition, Ef1 and its cell-free supernatant increased the transepithelial electrical resistance (TEER) of the enterocyte monolayer, thus strengthening the intestinal barrier against ETEC. These results may contribute to the development of therapeutic interventions using Ef1 in intestinal disorders of piglets.The probiotic Accoe (MAPK) , 15. Afte (MAPK) –18. The in China  and is ain China , and in pathogen . In thisEnterococcus faecium HDRsEf1 (Ef1) was isolated and identified by the Department of Veterinary Microorganisms & Immunity, Huazhong Agricultural University [iversity . Ef1 wasμm filter to remove any remaining bacteria. Cell-free supernatant equivalent to 1 × 108 CFU/mL was added to 1 mL antibiotic DMEM/F12 for the experiments described below.The cell-free supernatant from overnight cultures of Ef1 (S-Ef1) was prepared by centrifugation at 8000 rpm for 10 min at 4°C, followed by filtration through a 0.22 7 CFU/mL of Ef1, was added to 1 mL antibiotic-supplemented DMEM/F12 for the experiments described below.The EPS produced by HDRsEf1 were purified according to a procedure previously reported by Pan and Mei, with minor modifications . Briefly7 CFU/mL of Ef1, was added to 1 mL antibiotic-supplemented DMEM/F12 for the experiments described below.The protein produced by Ef1 was purified according to a procedure previously reported by Claes et al. with minor modifications . Briefly2 . The cells were cultured for at least 10 days, with the culture medium changed every other day.Porcine epithelial cells from the jejunum (IPEC-J2) were kindly donated by Professor Li Zili . The IPEC-J2 cells were seeded in cell culture flasks and cultured in DMEM/F12 medium supplemented with 10% foetal bovine serum , 1% penicillin-streptomycin , and 1% glutamine  at 37°C in a humidified atmosphere of 5% CO5 cells/mL were seeded into a 12-well plate and were cultured to allow differentiation. Adhesion assays were performed using fully differentiated IPEC-J2 cells (10 d postconfluence cultures). Bacteria were suspended in DMEM/F12 without antibiotics at concentrations of 5 × 107 CFU/mL (Ef1) and 5 × 107 CFU/mL (K88ac), and after the culture medium of IPEC-J2 was suck out, fresh medium containing the bacteria was added to wells and incubated for 1 h at 37°C in a 5% CO2 atmosphere. In the competition assay, Ef1 or S-Ef1 was added simultaneously with K88ac. For the exclusion assay, Ef1 or S-Ef1 was added first, and then 1 h later, K88ac was added and incubated for 1 h. For the displacement assay, K88ac was added first, and then 1 h later, Ef1 or S-Ef1 was added and incubated for 1 h. After incubation, nonadherent bacteria were discarded by washing thrice with sterile 1x PBS. The cells with adherent bacteria were lysed with 1 mL/well of Triton X  for 10 min in an ice-water bath. K88ac adhering to IPEC-J2 cells was serially diluted and spread onto MacConkey agar medium  for counting; Ef1 was also serially diluted and spread onto MRS to count the adherent bacteria. All experiments were performed three times independently.Approximately 5 × 102 Transwell®-COL collagen-coated membrane filters  to polarise the monolayer. IPEC-J2 cells were seeded at 1 × 106 cells per Transwell filter in 6-well tissue culture plates. TEER was measured every day after seeding, using the Millicell electrical resistance system . In order to avoid cell division, a high seed density was used to saturate the available area. At each measurement, duplicate values for at least two areas in each filter were obtained, and the results were expressed as Ω cm2. Cell monolayers with TEER levels above 4000 Ω cm2 were assumed to be fully polarised and were selected for the TEER test [8 CFU/mL) or S-Ef1 was added, preincubated for 2 h, and then washed with sterile 1x PBS (pH 7.4) thrice. Following this, 1 mL/well of K88ac (1 × 108 CFU/mL) was added as a stimulant for 12 h, and TEER of each sample was measured every 3 h. All experiments were performed three times independently.IPEC-J2 cells were seeded onto 4.2 cmEER test . Into a 5) were seeded into 12-well plates  and cultured at 37°C for 3 days in 5% CO2, and the cells were 100% confluent, and they were washed with sterile 1x PBS thrice, incubated with 5 × 107 CFU/well Ef1 or S-Ef1 for 2 h, and washed with sterile 1x PBS thrice. Then, 1 mL/well of K88ac (5 × 107 CFU/mL), 1 mL/well of IL-1β , and 1 mL/well of TNF-α  were added to each well and incubated for 2 h or 4 h. The bacteria, S-Ef1, IL-1β, and TNF-α were added in DMEM to IPEC-J2 cells.IPEC-J2 cells (105 cells/well) were seeded into 12-well plates  and cultured at 37°C for 3 days in 5% CO2, and the cells were 100% confluent and differentiated, and they were washed with sterile 1x PBS thrice. The washed cells were treated with 5 × 107 CFU/well Ef1 or S-Ef1 (heat-inactivated at 95°C for 30 min) for 2 h and washed with sterile 1x PBS thrice, and then 1 mL/well of K88ac (5 × 107 CFU/mL) was added and incubated for 2 h.IPEC-J2 cells (105 cells/well) were seeded into 12-well plates  and cultured at 37°C for 3 days in 5% CO2, and the cells were 100% confluent and differentiated, and they were washed with sterile 1x PBS thrice. The washed cells per well were treated with EPS or protein equivalent to culture volume containing 5 × 107 CFU Ef1 for 2 h and washed with sterile 1x PBS thrice, and then 1 mL/well of K88ac (5 × 107 CFU/mL), IL-1β (8 ng/mL), or TNF-α (200 ng/mL) was added and incubated for 2 h.IPEC-J2 cells . All experiments were performed three times independently.As described in p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗). All experiments were performed three times.Statistical evaluations were performed using the IBM SPSS-Statistics program for Windows, version 22 . Graphs were plotted with GraphPad Prism 5 software . Results are given as means ± SEM. The significance level for all analyses were set to Ef1 and K88ac were all able to adhere to IPEC-J2 cells after 1 h of incubation, and the adhesion ability of Ef1 is greater than that of K88ac . Coincubα, or IL-1β, and the expression of IL-8 was measured by qRT-PCR and ELISA.ETEC, which is a known pathogen and stimulator of IL-8, can damage IECs by modulating cytokines , 28. In 7 CFU/mL of HDRsEf1 clearly downregulated the IL-8 mRNA level, while K88ac strongly upregulated it . However, if the IPEC-J2 cells were pretreated by HDRsEf1 or S-Ef1 for 2 h, the IL-8 level was reduced by about one-third (p < 0.001) or one-half (p < 0.001), respectively  and 2.6-fold (p < 0.001) (α and IL-1β. Compared with TNF-α (200 ng/mL) and IL-1β (8 ng/mL) treatment alone, HDRsEf1 preincubation decreased the mRNA of IL-8 approximately 2-fold (p < 0.001) and 1.7-fold (p < 0.05), respectively, and S-Ef1 preincubation deceased the mRNA of IL-8 about 2.4-fold (p < 0.001) (p < 0.001)  , respect< 0.001)  and 1.4-< 0.001) , respect7 CFU/mL), TNF-α (200 ng/mL), or IL-1β (8 ng/mL) from 2 h to 4 h and then determined the IL-8 mRNA and protein levels. After 4 h of treatment with K88ac, TNF-α, IL-1β, the IL-8 mRNA, and protein levels increased significantly (p < 0.001), 9.2 times (p < 0.001), and 7.1 times (p < 0.001) (p < 0.05), 1237 pg/mL (p < 0.01), and 850 pg/mL (p < 0.001) versus the control at 244 pg/mL (p < 0.001), 7.8-fold (p < 0.05), and 1.7-fold (p < 0.001) , 626 pg/mL (p < 0.01), and 589 pg/mL (p < 0.05) , 4.7-fold (p < 0.001), 1.9-fold (p < 0.001) (p < 0.01), 621 pg/mL (p < 0.01), and 400 pg/mL (p < 0.01)  , respect44 pg/mL , respect< 0.001)  compared < 0.05)  versus K< 0.001) , and the < 0.01) , comparep < 0.001) and the mRNA levels of IL-8 were similar to that of the live HDRsEf1 and S-Ef1 (p > 0.05) (Pretreatment with heat-inactivated HDRsEf1 and S-Ef1 reduced the mRNA levels of IL-8 induced by K88ac ( > 0.05) . These r8 CFU/mL/well) for 2 h and then treated with K88ac (1 × 108 CFU/mL/well). The results showed that HDRsEf1 and S-Ef1 increased TEER at an early stage and that K88ac could significantly disrupt TEER in IPEC-J2. After stimulation with K88ac 3, 6, or 12 hours later, the levels of TEER decreased to 0.63 (p < 0.01), 0.52 (p < 0.01), or 0.12 (p < 0.01) relative to the original (1.0) (p < 0.05). HDRsEf1 had a long-term protective effect: 12 hours later, the epithelial barrier was functional (p < 0.05), while, with S- Ef1, the barrier was dysfunctional 3 hours later  . Howeverrs later .p < 0.001), TNF-α (p < 0.001), and IL-1β (p < 0.01) while the protein had no effect  effective in inhibiting Salmonella enterica serovar Typhimurium invasion of Caco-2 cells and in killing intracellular enteropathogens [ in vitro adherence of an ETEC strain to gangliotetraosylceramide molecules, which are physiological constituents of the mammalian intestinal epithelial surface [ S. choleraesuis serovar Typhimurium was due to an antimicrobial compound present in the culture supernatant of LB [For enteropathogens, attachment to IECs represents an essential step in establishing an infection. In pigs, ETEC is the most common etiologic agent of enteric diseases in the weaning period. ETEC infection induces a proinflammatory response in porcine IECs  and causathogens . Fujiwar surface , 38. Cocnt of LB . In thisDespite the known association between impaired intestinal barrier function, gastrointestinal disorders , 41, andα, IL-1β, or K88ac. The results of this study indicated that HDRsEf1 can modify IL-8 levels that are effective against enteropathogens and proinflammatory factors. Our data are in agreement with recent reports [ Lactobacillus rhamnosus L34 and L. casei L39 can inhibit Clostridium difficile-induced IL-8 production in IECs [ L. amylovorus and its supernatant inhibit TLR4 inflammatory signalling triggered by ETEC, and TLR2 is required for the suppression of TLR4 signalling [ L. delbrueckii have been shown to attenuate ETEC-induced inflammatory responses in porcine IECs, with TLR2/TLR4 playing a central role in the immunomodulatory action [Further, proinflammatory cytokines can be modulated by the microbiota in the gastrointestinal tract. Symbiotic bacteria, especially probiotic bacteria, can modify the expression of cytokines from epithelial cells , 49. Whe reports , 53 that in IECs . Some re in IECs , 56, sucgnalling . EPS of y action . Furthery action  showed tIn conclusion, we demonstrated that HDRsEf1 can adhere to IECs and inhibit IEC adhesion and proinflammatory action of ETEC K88ac. Specifically, it can fortify the epithelial cell layer and elicit anti-inflammatory responses in enterocytes. It is EPS rather than proteins in Ef1 cultural supernatant that do the probiotic effect, but the precise mechanisms of and the exact components of EPS that contribute to anti-inflammatory functions remain to be identified.


Spatial working memory (SWM) and the classical, tetanus-induced long-term potentiation (LTP) at hippocampal CA3/CA1 synapses are dependent on L-α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) containing GluA1 subunits as demonstrated by knockout mice lacking GluA1. In GluA1 knockout mice LTP and SWM deficits could be partially recovered by transgenic re-installation of full-length GluA1 in principle forebrain neurons. Here we partially restored hippocampal LTP in GluA1-deficient mice by forebrain-specific depletion of the GluA2 gene, by the activation of a hypomorphic GluA2(Q) allele and by transgenic expression of PDZ-site truncated GFP-GluA1(TG). In none of these three mouse lines, the partial LTP recovery improved the SWM performance of GluA1-deficient mice suggesting a specific function of intact GluA1/2 receptors and the GluA1 intracellular carboxyl-terminus in SWM and its associated behavior. Life Science Identifiers:tm1Rsp/JGria1                  Mus musculus >:< RRID:IMSR_JAX:019011>urn:lsid:<Sprengel>:< B6N.129-tm2RspGria2                     Mus musculus >:< RRID:MGI: 3612255>urn:lsid:<Sprengel>:< B6N.129-tm3RspGria2                     Mus musculus >:< RRID:EM: 09212>urn:lsid:<Sprengel>:< B6N.129-(Camk2a-cre)1GscTg                               Mus musculus >:< RRID:MGI:2181422>um:lsid:<Schutz>:<(Camk2a-tTA)1Mmay/JTg                     Mus musculus >:< RRID:IMSR_JAX:003010>urn:lsid:<Mayford>:<(tetO–lacZ-GFPGluA1(TG)8.1Rsp)Tg     Mus musculus >:< RRID:MGI:submitted>urn:lsid:<Sprengel>:<Changes in synaptic efficacy in the central nervous system are thought to underlie learning and memory. Activity-dependent and input-specific increases in excitatory postsynaptic responses, described as long-term potentiation (LTP) in hippocampal field recordings  have revealed an essential role for GluA1 in hippocampal LTP at Schaffer collateral/CA1 synapses. Thus, the CA3-to-CA1 LTP was strongly impaired in absence of GluA1  in the Morris Water Maze despite the absence of field-LTP  performance in rodents , a kainate receptor GluK1 or C-terminally truncated GluA1, was sufficient to restore LTP at mature CA1 synapses in absence of the endogenous AMPAR subunits  of the Heidelberg University. These guidelines adhere to the German Animal Welfare Act: Regulation for the Protection of Animals Used for Experimental or Other Scientific Purposes . Animal numbers for molecular and histological experiments were recorded under the protocol MPI/T-6/06; 15/08; 20/; 28/11. Genetic manipulations and behavioral experiments were licensed by the Regional Council in Karlsruhe, Germany (35-9185.81/G-4/02 and 35-9185.81/G-71/10). Efforts were made to minimize the number of animals used.Gria1−/−/2FbΔ, Gria1−/−/2QFb, and Gria1−/−/Tg8.1 mice the following gene-targeted and transgenic mouse lines were used as founder lines:For the generation of Gene-targeted mice:Gria1−/− 1Gsc; Mantamadiotis et al., Tg8.1 (TgtetO-lacZ-GFPGluA1(TG)8.1Rsp1Mmay: Gria1+/−/Gria2lox+/X Gria1+/−/Gria2lox+//TgCre4. Littermates with the genotypes Gria1+/+/2+/+, Gria1+/+/2lox+/ or Gria1+/+/22lox were used as wild-type controls, Gria1−/−, Gria1−/−/TgCre4, Gria1−/−/2lox+/ or Gria1−/−/2lox/lox were used as GluA1 deficient mice in behavioral experiments.−/−/2QFbGria1(Gria1−/−/Gria2neo+//TgCre4): Gria1+/−/Gria2neo+/ X Gria1+/−/TgCre4. Littermates with the genotypes Gria1/2+/+ or Gria1+/+/TgCre4 were used as wild-type controls, Gria1−/−/TgCre4 or Gria1−/−/2+/+ were used as GluA1 deficient mice in behavioral experiments.−/−/Tg8.1Gria1 (TgaCaMKII-tTA/TgtetOhbox−lacZ-GFPGluA1(TG)8.1Rsp):(Gria1+/−/TgaCaMKII-tTA X Gria1+/−/TgtetO-lacZ-GFPGluA1(TG)8.1, 3′intro3 (5′-CTG CCT GGG TAA AGT GAC TTG G-3′), 2X1Lox-pz (5′-CAC TCA CAG CAA TGA AGC AG-3′), Gria1+: 191 bp and Gria1−: 265 bp.Gria2neo+/: MH60 (5′-CAC TCA CAG CAA TGA AGC AGG AC-3′), MH53a (5′-GAA TGT TGA TCA TGT GTT TCC CTG-3′) and MH117 (5′-GTT CGA ATT CGC CAA TGA CAA GAC G-3′), Gria2+: 500 bp and Gria2neo: 400 bp.Gria22lox: VM12 (5′-GCG TAA GCC TGT GAA ATA CCT G-3′) and VM10 (5′-GTT GTC TAA CAA GTT GTT GAC C-3′), Gria2+: 250 bp and Gria2lox: 350 bp.Tg8.1): VM-70 (TGG GAG CCA CAG GAT AAA AGC) and VM-72 (GTG AGC CAA GAT TGT GCC ACT GC) to amplify a 286 bp DNA fragment.TgCre4: rspCre1 (5′-ACC AGG TTC GTT CAC TCA TGG-3′) and rspCre2 (5′-AGG CTA AGT GCC TTC TCT ACA C-3′) to amplify a 200 bp DNA fragment.TgaCaMKII-tTA: Ca25: GCT CAG AAG CCC CAA GCT CG and CAs25as: CAG CGC CTA ACT CTG GAC AC and Casli 3: TAA GCA GCT CTA TGC GCT GTT A to amplify a PCR fragment in wild-type of 380 bp and transgenic mice of 500 bp , anti-GluA1  and anti-GluA2  in combination with secondary anti-mouse (RRID:AB_2336176) and anti-rabbit (RRID:AB_2313567) antibodies coupled to horseradish-peroxidase  or with biotinylated secondary antibodies  and ABC-peroxidase kit  were used.Coronal 70–100 μm thick vibratome sections were analyzed using different primary antibodies as described , anti-GluA2 , anti-GluA3 , anti-GluA4 , anti-GluN1 , anti-β actin ; secondary goat anti-rabbit (RRID:AB_2336198) and goat anti-mouse coupled to horseradish-peroxidase . Data are presented as mean ± SEM. Western blot quantification was statistically evaluated by analysis of variance (ANOVA) measurements followed by Holm-Sidak's multiple comparison and Bonferroni post-hoc tests .Mouse brains were removed and both hippocampi were isolated. Total protein was prepared and immunoblots were performed as described . Patch pipettes were pulled from borosilicate glass capillaries and had resistances of 4–7 MΩ when filled with (in mM) 125 Cs-gluconate, 20 CsCl, 10 NaCl, 10 HEPES, 0.2 EGTA, 4 MgATP, 0.3 Na3GTP, 100 μM spermine, and 2.5 mM QX-314 . All chemicals were obtained from Sigma. Series resistances and input resistances were continuously monitored by measuring peak and steady-state currents in response to hyperpolarizing pulses . Liquid junction potentials were corrected. Synaptic currents were activated between −70 and +40 mV in 10 mV steps by stimulating the Schaffer collateral/commissural fibers in str. radiatum 150 μm away from the CA1 cell body with a glass electrode filled with 1 M NaCl. AMPAR currents were recorded in presence of 50 μM D-2-amino-5-phosphonopentanoic acid , 10 μM bicuculline methiodide (Sigma) and 1 μM CGP 55845 (Tocris). Single traces were analyzed and illustrated. The rectification index (RI) is given as the current ratio at +40 and −60 mV. Data are presented as mean ± SEM. Statistical significance was evaluated by a two-tailed, unpaired Student's t-test.Brains were removed from deeply anesthetized mice  and transverse hippocampal slices (250 μm) were prepared and incubated for 30 min at 37°C in artificial CSF (ACSF) containing (in mM): 125 NaCl, 25 NaHCOstr. radiatum and in the str. oriens. The former was used to induce LTP, whereas the latter activated the control pathway. Excitatory postsynaptic currents (EPSCs) were elicited by activation of the two pathways (0.2 Hz) and were recorded for 20 min at –70 mV after the LTP-induction. The following intra- and extra-cellular solutions were used: Intracellular (in mM): 120 CsGluconate, 10 CsCl, 8 NaCl, 10 HEPES, 10 phosphocreatine, 0.2 EGTA, 4 MgATP, 0.3 NaGTP. The pH was set to 7.24 with CsOH and osmolarity was analyzed (295–310 mOsm). Extracellular (in mM): 124 NaCl, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 4 MgSO4, 4 CaCl2, 10 glucose. All chemicals were obtained from Sigma. Statistical analysis was done by a two-tailed paired Student's t-test.Pairing-induced LTP was induced by pairing low frequency stimulation  with postsynaptic depolarization to 0 mV for 3 min as published in Chen et al. . Monopolt-test. LTP levels between genotypes were evaluated by linear mixed model statistical analysis .Potentiation of hippocampal field excitatory postsynaptic potentials (EPSPs) was induced by tetanic stimulation as previously published. In all these studies Vidar Jensen and Øivind Hvalby performed the experiments under the same conditions and at the same E-Phys. setups .SWM was administrated in the rewarded alternation task on an elevated T-maze .Acquisition of SRM was performed with mice kept on a strict food diet (remain to 85–90% of the starting body weight) on an elevated Y-shaped maze with prominent extra-maze cues as previously described  was achieved in Gria1−/−/2FbΔ and Gria1−/−/2QFb mice either by Cre-mediated deletion of the Gria22lox gene . Gria1−/−/2QFb mice were also viable, but the epileptic phenotype observed in mice with forebrain-specific, heterozygous GluA2(Q) expression  and Gria1−/−/2QFb mice (61.7 ± 4.5%) compared to control (98.7 ± 5.4%) and Gria1−/− mice . In Gria1−/−/2QFb mice GluA3 levels seemed to be more reduced. However, the difference between GluA3 levels in Gria1−/−/2FbΔ and Gria1−/−/2QFb did not reach statistical significance (p = 0.22). The levels of the NMDAR subunit GluN1  were not altered. Similarly, the amount of the GluA4 subunit, which is not expressed in mature hippocampal pyramidal neurons, was unchanged .To quantify and to visualize the expression of AMPAR subunits, we determined the hippocampal expression pattern of GluA1–3. As expected from the Cre expression pattern of e Figure . The nor2+-permeable AMPARs in hippocampal pyramidal cells, we performed whole-cell recordings in acute hippocampal slices of Gria1−/−/2FbΔ and Gria1−/−/2QFb mice. In accordance with previous studies of homomeric GluA3 AMPARs  compared to Gria1−/− . In Gria1−/−/2QFb, the presence of the higher conducting GluA2(Q)-containing AMPARs . Importantly, both genotypes expressed Ca2+-permeable AMPARs in CA1 pyramidal cells, as indicated by rectification indices (RIs) of current-voltage relationships , which is characteristic of Ca2+-permeable AMPARs lacking the GluA2 subunit  can be explained by the presence of two Gria2 alleles (Gria2 and Gria2ECSΔ) leading to a mixed AMPAR population containing Ca2+-permeable GluA2(Q) and Ca2+-impermeable GluA2 receptors.In order to show that the remaining GluA3 and the activated GluA2(Q) subunits form Cas Figure . In wildGria1−/−/2QFb mice by a small but significant amount of NMDAR-independent LTP  measured in the presence of the NMDAR antagonist D-AP5  mice  in voltage-clamp, whole cell recordings of hippocampal CA1 neurons elicited a robust and long-lasting potentiation of excitatory postsynaptic currents (EPSCs) compared to the un-paired control pathway in str. oriens . Consistent with previous observations . Moreover, expression of GluA2(Q) in Gria1−/−/2QFb mice enabled potentiation of CA1 EPSCs  which was similar to pairing-induced LTP of control mice  and in Gria1−/−/2QFb  we could also monitor LTP in e Figure . Field eGria1−/− mice that express a transgenic, PDZ motif-truncated and GFP-tagged GluA1(TG) mutation . In these mice the αCaMKII promoter-driven transgene TgaCaMKII-tTA , but was still significantly reduced (p < 0.05) when compared to LTP of wild-type mice -expressing GluA1 knockout mice  developed regular levels of LTP but showed strong SWM deficits  while Gria1−/− performed not different to chance level (53.4 ± 1.6) as reported  = 22.51, p < 0.0001] and block , but any genotype-by-block interaction . Post-hoc Student-Newman-Keuls comparison identified significant differences (p < 0.05) for all three mouse models  vs. control mice while mutants did not differ among each other. In addition, one sample t-test to the theoretical mean of 50% SWM performance (chance level) revealed significant values (p < 0.05) for control mice while all mice of the GluA1-deficient mouse lines did not perform differently from chance level.Regardless of the activated expression of endogenous CaGria1−/−/2FbΔ mice were also tested to learn a fixed location of an arm in a Y-maze in 10 blocks of 10 trials each , supporting the finding of Gria1−/− mice that the SWM is not a prerequisite for the formation of SRM  indicating a specific role of GluA2 for certain behaviors.h Figure . Gria1−/Gria1 and Gria2 genes to modulate hippocampal AMPAR expression in GluA1-deficient mice. The cell-type specific modulation of AMPARs was achieved by inactivating a floxed Gria2 gene, by activating a hypomorphic Gria2neo gene and by expressing a transgenic GFP-tagged-GluA1(TG) in principal forebrain neurons of GluA1 knockout mice. In the three different mouse lines—Gria1−/−/2FbΔ, Gria1−/−/2QFb, and Gria1−/−/Tg8.1—the remaining AMPAR levels and the ratios of Ca2+-permeable and Ca2+-impermeable AMPARs is very different in principal neurons of the hippocampus.In our study we used genetically modified Gria1−/−/2FbΔ mice, the GluA3 level, was about 25% lower compared to GluA3 levels of wild-type mice, where GluR3 subunits already represent only 10% of the AMPAR subunits , the fall in GluA3 expression was less pronounced than in Gria1−/−/2FbΔ mice even though the difference reached no statistical difference (p = 0.22). This might suggest that AMPARs containing only Glutamine (Q) in the pore-forming segment  at homologous position. Similarly the two-fold reduction of GluA2 levels in Gria1−/−/2QFb mice is less pronounced when GluA1 is present in Gria2QFb  mice, as demonstrated in an earlier study —the hallmark of Ca2+-permeable AMPARs  in AMPAR assemblies could also be monitored by the formation of synaptic Ca2+-permeable AMPARs, as shown by the small but significant shift of the RI compared to the RI monitored in wild-type mice; the AMPAR-mediated current amplitude was similar to wild type.The immunohistological analysis of coronal brain slices confirmed the absence and reduced GluA2 expression in Gria1−/−/2FbΔ and Gria1−/−/2QFb mice was sufficient for the induction and expression of pairing-induced and field-LTP in GluA1-deficient mice. However, the different amount of AMPARs affected the potentiation level. The GluA3-containing AMPARs of Gria1−/−/2FbΔ mice showed slightly lower LTP levels compared to the partial LTP rescue of Gria1−/−/2QFb mice. A partial recovery of field-LTP in Gria1−/− mice was also achieved by the transgenic GFP-GluA1(TG) subunit in Gria1−/−/Tg8.1 mice confirming that the GluA1-PDZ domain is dispensable for LTP  was associated with strong SWM impairment (Shimshek et al., Despite the partially restored hippocampal LTP in our three mouse lines, the SWM performance of all three lines remained at the chance level in the T-maze task. The lower amplitudes of LTP are unlikely to be the main reason for the failure to rescue the SWM impairment of GluA1 knockout mice. As we described earlier a partial LTP rescue with similar amplitudes obtained by the transgenic GFP-tagged-GluA1 expression was sufficient to improve the SWM performance in DS, TB, LL, and RS designed, generated, and molecularly analyzed the mouse lines. VJ, BS, and GK performed and analyzed the electrophysiological experiments. VM and DS performed the behavioral experiments. RS, DS, and TB wrote the manuscript.DS and BS are currently employed by Novartis Pharma AG, Basel, Switzerland. However, this work was completed whilst DS and BS were employed at the Max Planck Institute for Medical Research. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


E)-N′-[(1H-indol-3-yl)methylidene]isonicotinohydrazide molecules and (E)-N-methyl-2-[1-(2-oxo-2H-chromen-3-yl)ethylidene]hydrazinecarbothioamide molecules form a tape structure and a helical chain structure, respectively, through hydrogen bonds.In the two title Schiff base derivatives, the ( 15H12N4O·C2H6O (1·EtOH) and C13H13N3O2S (2), were determined at 110 and 100 K, respectively. In the crystal of compound 1·EtOH, the (E)-N′-[(1H-indol-3-yl)methylidene]isonicotinohydrazide and ethanol molecules are linked by O—H⋯O, N—H⋯O and N—H⋯N hydrogen bonds, forming a tape structure running along the b-axis direction. The tapes are weakly linked via a C—H⋯N interaction. In the crystal of compound 2, (E)-N-methyl-2-[1-(2-oxo-2H-chromen-3-yl)ethylidene]hydrazinecarbothioamide molecules are linked via N—H⋯O and C—H⋯O hydrogen bonds, forming a helical chain along the b-axis direction. The chains are further linked into a layer expanding parallel to (102) through C—H⋯S interactions.The crystal structures of two title Schiff base derivatives, C The mean plane of the central chain C9/N2/N3/C10/O1 in 1 makes dihedral angles of 6.91 (12) and 42.71 (13)°, respectively, with the C1–C8/N1 ring system and the pyridine C11–C15/N4 ring. In molecule 2, the dihedral angle between the C1–C9/O1 ring system and the mean plane of the C10/N1/N2/C12/N3/C13 chain is 30.36 (9)°.The molecular structures  and C(7) chain motifs. The C—H⋯N interaction generates a C(8) chain. Atom O1S of the ethanol molecule acts as a donor in forming the O—H⋯O hydrogen bond with atom O1, which acts as a double acceptor.The crystal packing of s Table 1, which lon Fig. 3. The tapn Table 1. In the 2, the crystal packing features N—H⋯O, C—H⋯O and C—H⋯S interactions . Atoms N2 and C11 act as donors to the double acceptor O2, generating C(7) and C(6) chains, respectively. As a result of these two hydrogen bonds, an C(11) and C(7) chains, respectively.In s Table 2. The molon Fig. 4. The chaet al., 20161 and 2 revealed several related Schiff base derivatives, including those with refcodes ADEKAW, ACIPIN, ADEZAL02 and APAQEP reported by Qiu et al.  with nicotinic acid hydrazide  in ethanol. The reaction mixture was then refluxed on a water bath for 5 h and poured into crushed ice. The corresponding solid Schiff base that formed was filtered, washed several times with distilled water and dried under vacuum. The compound was recrystallized from an ethanol–chloroform (1:3) solvent mixture, yielding the ethanol solvate compound, 1·EtOH. Similarly, compound 2 was synthesized from equimolar amounts of 3-acetyl-2H-chromen-2-one  with N-methylhydrazinecarbothioamide  in ethanol. Compound 2 was also recrystallized from an ethanol–chloroform (1:3) solvent mixture.Compound Uiso(H) = 1.2 or 1.5Ueq(parent atom). For 1·EtOH, the methylene H atoms of the ethanol solvent molecule were refined independently under strong bond-length and angle restraints using DFIX to avoid a large residual electron-density peak near the methylene C atom caused by the usual riding treatment of the H atoms. In 2, TWINABS was employed to correct the data for absorption effects, as well as to separate hkl files for the domains with major and minor components; the twin ratio was observed to be 91:9. In the refinement, only the data of the major domain were used.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S205698901700411X/is5471sup1.cifCrystal structure: contains datablock(s) Global, 2, 1.EtOH. DOI: 10.1107/S205698901700411X/is54711.EtOHsup4.hklStructure factors: contains datablock(s) 1.EtOH. DOI: 10.1107/S205698901700411X/is54712sup5.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S205698901700411X/is54711.EtOHsup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S205698901700411X/is54712sup5.cmlSupporting information file. DOI: 1537754, 1537753CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N-oxide complexes are presented.The synthesis and structures of three manganese(II) pyridine  N-oxide complexes have been synthesized from the reaction of manganese(II) chloride with either pyridine N-oxide (PNO), 2-methylpyridine N-oxide (2MePNO) or 3-methylpyridine N-oxide (3MePNO). The compounds were synthesized from methanolic solutions of MnCl2·4H2O and the respective N-oxide, and subsequently characterized structurally by single-crystal X-ray diffraction. The compounds are catena-poly[[aquachloridomanganese(II)]-di-μ-chlorido-[aquachloridomanganese(II)]-bis(μ-pyridine N-oxide)], [MnCl2(C5H5NO)(H2O)]n or [MnCl2(PNO)(H2O)]n (I), catena-poly[[aquachloridomanganese(II)]-di-μ-chlorido-[aquachloridomanganese(II)]-bis(μ-2-methylpyridine N-oxide)], [MnCl2(C6H7NO)(H2O)]n or [MnCl2(2MePNO)(H2O)]n (II), and bis(μ-3-methylpyridine N-oxide)bis[diaquadichloridomanganese(II)], [Mn2Cl4(C6H7NO)2(H2O)4] or [MnCl2(3MePNO)(H2O)2]2 (III). The MnII atoms are found in pseudo-octahedral environments for each of the three complexes. Compound I forms a coordination polymer with alternating pairs of bridging N-oxide and chloride ligands. The coordination environment is defined by two PNO bridging O atoms, two chloride bridging atoms, a terminal chloride, and a terminal water. Compound II also forms a coordination polymer with a similar metal cation; however, the coordination polymer is bridged between MnII atoms by both a single chloride and 2MePNO. The distorted octahedrally coordinated metal cation is defined by two bridging 2MePNO trans to each other, two chlorides, also trans to one another in the equatorial (polymeric) plane, and a terminal chloride and terminal water. Finally, complex III forms a dimer with two bridging 3MePNOs, two terminal chlorides and two terminal waters forming the six-coordinate metal environment. All three compounds exhibit hydrogen bonding between the coordinating water(s) and terminal chlorides.Three manganese(II)  N-oxides to facilitate organic oxo-transfer reactions has been well documented over the years   complexes. Notably, we used the ligands pyridine N-oxide, 2-methylpyridine N-oxide, and 3-methylpyridine N-oxide to study the impact of substitution of the pyridine on the two- and three-dimensional solid-state structures. The pyridine N-oxide (PNO) and 2-methylpyridine N-oxide (2MePNO) complexes form coordination polymers with subtle differences. The 3-methylpyridine N-oxide (3MePNO), however, forms a dimeric complex.In this context, we report the synthesis and solid-state structures of three pyridine I exhibits the repeating motif of [MnCl2(PNO)(H2O)]n and crystallizes in the triclinic space group PII atom is a distorted octahedron, with the equatorial atoms being two bridging chlorides alternating with two bridging pyridine N-oxide (PNO) molecules  Å, whereas the Mn1—O1vii bond length is slightly longer at 2.182 (3) Å for the bridging PNO . The bridging chlorides are found to have Mn—Cl2 distances of 2.5240 (19) and 2.532 (19) Å, respectively. Axially, the water is located 2.250 (3) Å from the MnII cation and the terminal chloride is at 2.479 (2) Å. The bond angles around the equator are severely compressed at the two bridging N-oxides, with the O1—Mn1—O1i angle observed at 72.03 (10)°. The remaining three angles are found to all be similar at 95.58 (7) (Cl2—Mn1—Cl2i), 96.80 (8) (O1—Mn1—Cl2), and 94.69 (9)° (O1vii—Mn1—Cl2vii). Axially, the bond angle from the water through manganese(II) and the terminal chloride (O2—Mn1—Cl1) is nearly linear at 177.36 (8)°.Complex ll Fig. 1. The cooes Fig. 2. In the II, [MnCl2(2MePNO)(H2O)]n, posseses a metal environment similar to complex I and crystallizes in the orthorhombic space group P212121. The major difference in structure II is in the bridging network, where the chlorides and N-oxides are trans to one another rather than cis as in I  bond length of 2.170 (6) Å, with a Cl1—Mn1—O1 bond angle of 84.37 (19)°. The bond angle across the Cl atoms, Cl1—Mn1—Cl1viii, is 174.02 (5)° and across the O atoms of 2MePNO, O1—Mn1—O1ix, is 173.12 (6)°; a slight compression is observed across the bridges 2, crystallizes in the triclinic PN-oxide bridge, and the other equivalents are axial in the pseudo-octahedral geometry around the MnII atoms. The Mn1—Cl1 and Mn1—Cl2 bond lengths are 2.4601 (5) and 2.4903 (19) Å, respectively, with a Cl1—Mn1—Cl2 bond angle of 98.32 (4)°. The bridging N-oxide is at a distance of 2.1791 (18) Å from Mn1—O1, with an O1—Mn1—O1vii bond angle of 71.86 (7)° . The Mn1—O2(water) and Mn1—O3(water) bond lengths are 2.245 (2) and 2.1696 (17) Å, respectively, with an O2—Mn1—O3 bond angle of 85.83 (7)°.The dimeric complex er Fig. 5. The 3-mI and IIversus the dimer in III is likely due to the steric influence of the methyl group in the 3-position in 3MePNO and the core constituents. One can define the Mn2 ‘N-oxide diamond core’ in each of the structures as follows: I is alternating Mn2Cl2 and Mn2O2 (oxygen bridges via PNO) cores, II is Mn2ClO (oxygen bridge via 2MePNO) and III Mn2O2 (oxygen bridges via 3MePNO). In I, the unsubstituted pyridine N-oxide group does not generate as much steric strain, allowing for polymer formation. In II, the core is formed to permit alternating up and down pyridine N-oxides with the 2-methyl substituents also facing in opposite directions. This limits the steric interactions and the N-oxide slightly tilts out of the polymeric core line to allow the methyl group to effect less steric interactions. In III, the methyl group appears to inhibit polymer formation due to the position of this bulky substituent. Subsequently, when the polymer is not formed, an extra water molecule is required to fill the sixth coordination site on the metal cation occupied by a bridging atom in I and II.The formation of the polymeric structure in I forms a coordination polymer of alternating bis-bridges of two chlorides and two pyridine N-oxides in the a-axis direction  is located down the bridge to the terminal chloride (Cl1), on the adjacent MnII atom, and the O2—H2A⋯Cl1i distance is 2.53 (2) Å. The other hydrogen-bond interaction (H2B) is across to the next polymeric chain with Cl1; the O2—H2B⋯Cl1ii distance is 2.52 (3) Å , II has a single 2-methylpyridine N-oxide and a single chloride in each bridge. Similar to I, the hydrogen-bonding interactions are to a terminal chloride (Cl2) on the adjacent MnII atom. There are two observed interactions, viz. O2—H2A⋯Cl2iii with a distance of 2.49 Å and O2—H2B⋯Cl2iv with a distance of 2.26 Å  Å.Complex  I Fig. 4. HoweverIII does not form a coordination polymer but is observed in the solid state as a dimer with two water molecules for each MnII atom   Å] and O3—H3A⋯Cl2vi [distance 2.28 (2) Å]  Fig. 5. The aroet al., 2016N-oxides bound to manganese returned 87 entries. Similar N-oxides with simple counter-ions in the list include 4,4′-dipyridine N,N′-dioxide [FIVHAU  was dissolved in a minimal amount of methanol, approximately 10 ml. Two stoichiometric equivalents of the appropriate N-oxide were also dissolved in approximately 20 ml of methanol . The solutions were stirred for approximately 10 min; during each reaction, a brown solution was observed upon mixing. The reaction solution was then allowed to sit and brown crystals were grown by slow evaporation in the near quantitative yields reported below based on the manganese(II) chloride starting material. The FT–IR spectra of the complexes all exhibit broad absorbances in the 3400–3000 cm−1 region due to the ν(O—H) of the coordinating water molecules, as well as the characteristic ν(N—O) of the N-oxide pyridyl moiety in the 1250–1150 cm−1 region noted previously Cl2·H2O, yield 0.215 g (90.3%). Selected IR bands : 3364 , 3235 , 3068 , 1660 (w), 1471 (w), 1214 (m), 1205 (m), 1023 (s), 831 (s), 780 (s), 674 (s), 556 (s). Elemental analysis for MnCl2C5H7NO2, calculated (%): C 25.13, H 2.95, N 5.86; found (%): C 25.22, H 2.96, N 5.87.Compound II, Mn(2MePNO)Cl2·H2O, yield 0.227 g (87.9%). Selected IR bands : 3410 , 3247, 3073, 1716 (w), 1619 (w), 1578 (w), 1421 (m), 1264 (m), 1154 (m), 1029 (s), 831 (s), 799 (s), 684 (s), 584 (s). Elemental analysis for MnCl2C6H9NO2, calculated (%): C 28.48, H 3.59, N 5.53; found (%): C 28.75, H 3.53, N 5.28.Compound III, Mn2(3MePNO)2Cl4·4H2O, yield 0.231 g (89.5%). Selected IR bands : 3374 , 3251 , 3094 , 1663 (w), 1614(w), 1492 (m), 1261 (m), 1164 (m), 1019 (s), 946 (s), 750 (s), 672 (s). Elemental analysis for Mn2Cl4C12H22N2O6, calculated (%): C 26.59, H 4.09, N 5.16; found (%): C 26.53, H 4.04, N 5.21.Compound Uiso(H) = 1.2Ueq(C) or Uiso(H) = 1.5Ueq(C) for C(H) and CH3 groups, respectively. In order to ensure chemically meaningful O—H distances for the bound water molecules in compound I, the H2A—O2 and H2B—O2 distances were restrained to a target value of 0.84 (2) Å  = 1.5Ueq(O) using an AFIX 7 command, and in compound III, H2A—O2, H2B—O2, H3A—O3, and H3B—O3 were restrained using DFIX as for compound I. A rotating-group model was applied for the methyl groups. Structure refinement of II exhibits inversion twinning. Several crystals were tried and the centrosymmetric space group Pnma was tested. In all cases, there was a significant reduction in the R value for the inversion twinning P212121 solution.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989017012038/zl2709sup1.cifCrystal structure: contains datablock(s) I, II, III. DOI: 10.1107/S2056989017012038/zl2709Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017012038/zl2709IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989017012038/zl2709IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 1570001, 1570000, 1569999CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Page 1, column 1, lines 26 and 27: “silencing mediator for retinoic acid and thyroid hormone receptor” should read “single-molecule real-time.”Volume 3, no. 2,


Z′ varying from ½ to 4, depending on the nature of a single functional group.Four bromoarenes are characterized by different contents for their asymmetric units, with  X placed para to the Br atom: X = CN , (1), X = NO2 , (2), X = NH2 , (3) and X = OH , (4). The content of the asymmetric unit is different in each crystal, Z′ = ½ (X = CN), Z′ = 1 (X = NO2), Z′ = 2 (X = NH2), and Z′ = 4 (X = OH), and is related to the molecular symmetry and the propensity of X to be involved in hydrogen bonding. In none of the studied compounds does the crystal structure feature other non-covalent interactions, such as π–π, C—H⋯π or C—Br⋯Br contacts.The crystal structures of four bromoarenes based on 2,6-dimethylbromobenzene are reported, which are differentiated according the functional group  Another structural invariant over the studied series is the minimization of steric crowding effects between the Br atom and the methyl groups in ortho positions. The methyl groups are systematically rotated in such a way that the C—Br bond is staggered with a CH2 fragment of the methyl group. As a consequence, the endocyclic angle at the Br-bearing C atom is always the largest one in the benzene ring, varying from 121.8 (3)° in (3) to 123.9 (4)° in (1).No unusual bond lengths or angles are observed in the four molecules Figs. 1 ▸ ▸. FZ′ are obtained for the four compounds. Molecule (1) (X = CN) has the highest potential molecular symmetry, Cv2, assuming a linear C—C≡N group. Omitting H atoms, this symmetry is actually reached, with the C—Br and C—C≡N fragments lying on the mirror plane in space group P21/m , with X = NO2, the latent symmetry Cv2 is broken because the nitro group is tilted slightly with respect to the benzene ring by an angle of 13.0 (4)°. For this crystal, Z′ = 1 in space group P3) and (4), which are isoelectronic molecules , despite the molecular symmetry being close to Cv2, the asymmetric units contain more than one molecule: Z′ = 2 for (3)   Fig. 3 and Z′ =4) Fig. 4, in spacZ′, may be rationalized on the basis of two key parameters. First, a higher molecular symmetry obviously favours the crystallization of low Z′ crystals, as in (1). This has been observed in many symmetrically substituted benzene derivatives, for example, in 4-bromo-benzonitrile in space group Cm , for which the molecular symmetry is lowered to C1. Secondly, the introduction of efficient donor groups for hydrogen bonding, such as NH2 and OH groups, is an enabling factor for crystal structures having Z′ > 1, as observed for (3) and (4). A search in the organic subset of the CSD  is featureless regarding the packing of the molecules. No short contacts such as halogen bonds are formed, and π–π interactions are insignificant, the shortest separation between benzene ring being defined by cell translations along the short cell axis, a = 4.0382 (1) Å.As expected, compound (2), two pairs of weak C—H⋯O hydrogen bonds link the molecules to form two centrosymmetric first-level ring motifs of 2R2(10), with the participation of the nitro group as acceptor , slipped π-stacking interactions are insignificant, the benzene-to-benzene distance being, again, determined by the cell axis a = 4.0502 (5) Å.For  and (4) are isoelectronic, they present different crystal structures. This is because their donor groups for hydrogen bonding are of a different nature: the N—H bond is a poorer donor compared to the O—H bond, on the basis of the polarity of these bonds, estimated with the differences of electronegativity χN − χH = 0.84 and χO − χH = 1.24 . Moreover, the NH2 group is potentially involved in two hydrogen bonds, while the OH group is expected to form a single, stronger contact, at least as long as bifurcated hydrogen bonds are not considered.Although compounds (3) and (4) have a supramolecular structure based on chains oriented along a screw 21 axis , two discrete contacts D(2) are formed between the two independent molecules s Table 2. These c0] Fig. 6, top.D and C motifs appears in (4), this time starting from a Z′ = 4 asymmetric unit: three discrete motifs D(2) are formed within the asymmetric unit, and a fourth D(2) motif connects the first independent molecule with a symmetry-related molecule in the crystal (Table 34) are much more efficient than those observed in (3): all O—H⋯O bonds have short H⋯O distances of ca 1.9 Å and O—H⋯O angles are close to 180° –(4) include π–π or C—H⋯π contacts, nor C—Br⋯Br halogen bonds. The arrangement of the molecules in the crystal over the studied series of compounds is thus mainly determined by the absence of, the presence of weak, or strong hydrogen bonds, respectively, in (1), (2) and (3), or (4).It is worth noting that none of the observed 1D supramolecular structures in .Polysubstituted benzene systems are ubiquitous in the crystallographic literature. Limiting a survey to 2,6-dimethylbromobenzene, only two derivatives closely related to the series we have studied may be found, with 3) was purchased from Oakwood Chemical Co. and was the starting material for the synthesis of (2) by oxidation with m-CPBA, and (1) and (4) via a Sandmeyer reaction. Single crystals of (3) were obtained by slow evaporation of a CH2Cl2 solution.Compound  in water (5 ml) was added dropwise to a suspension of 4-bromo-3,5-dimethylaniline  in aqueous HCl , and water (2 ml) at 273 K. The mixture was stirred at 273 K for 30 min and then neutralized with NaHCO3. Separately, a solution of CuCN , and KCN  in water (10 ml) was heated at 343 K. This solution was added dropwise to the diazotization solution previously prepared. The mixture was kept at 343 K for 30 min with stirring and then cooled at room temperature. The product was extracted with toluene (3 × 30 ml). The combined organic layers were dried over anh. Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography  to obtain compound (1) as orange needles ; m.p. 408–410 K; IR: 3022 (C—H Ar), 2354 (C≡N), 1498 (C=C) cm−1; 1H NMR : δ 7.34 , 2.44  p.p.m.; 13C NMR  δ: 140.0, 133.2, 131.1, 118.4, 110.7, 23.8 p.p.m.; GC–MS (EI): m/z = 209 (100%) [M+], 211 (97%) [M+ + 2] amu. Single crystals suitable for X-ray analysis were obtained by slow evaporation of a CH2Cl2 solution.Compound (1) was prepared by modification of the reported procedure (Xu 2) was prepared by modification of the reported procedure  was heated at 323 K for 2 h. After cooling at room temperature, the precipitate was filtered off and the liquid phase was washed with NaOH . The organic layer was dried over anh. Na2SO4 and concentrated under reduced pressure. The residue was dissolved in glacial acetic acid (10 ml), and a solution of H2O2  and glacial acetic acid (5 ml) was added at room temperature. Then, conc. HNO3 (0.5 ml) was slowly added and the mixture was heated to 363 K for 4 h. After cooling, the crude was treated with water (50 ml), and was extracted with CH2Cl2 (3 × 50 ml). The combined organic layers were dried over anh. Na2SO4 and concentrated under reduced pressure. The crude was purified on a silica gel column chromatography (petroleum ether) to give compound (2) as bright-yellow crystals ; m.p. 478–483 K; IR: 2988 (C—H Aliph), 1558, 1340 (N—O) cm−1; 1H NMR : δ 7.92 , 2.51  p.p.m.; 13C NMR : δ 146.3, 140.1, 134.8, 122.5, 24.1 p.p.m.; GC–MS (EI): m/z = 229 (100%) [M+], 231 (97%) [M++2] amu. Crystals suitable for single crystal X-ray diffraction were obtained by slow evaporation of an ether solution.Compound (4): A solution of 4-bromo-3,5-dimethylaniline  in conc. H2SO4 (25 ml) and water (5 ml) was cooled to 273 K. Then a solution of NaNO2  in water (10 ml) was added dropwise under stirring. After additional 30 min the solution was refluxed for 30 min. The mixture was cooled and extracted with EtOAc (3 × 50 ml). The combined organic phases were dried over anh. Na2SO4 and concentrated under reduced pressure. The crude was purified by silica gel column chromatography  to provide the product (4) as pale-orange crystals ; m.p. 386–388 K; IR: 3620 (O—H), 2987 , 1590 (C=C Ar), 1120 (C—O) cm−1; 1H NMR : δ 6.57 , 4.99 , 2.34  p.p.m.; 13C NMR : δ 153.9, 139.5, 118.3, 115.2, 23.8 p.p.m.; GC–MS (EI): m/z = 200 (100%) [M+], 202 (97%) [M+ + 2] amu. Crystals suitable for diffraction were obtained by slow evaporation of an EtOAc solution.Preparation of (3) decomposes after a few minutes under Mo Kα irradiation, but is stable for hours under Cu Kα irradiation. For compound (3), H atoms of NH2 groups were located in a difference Fourier map and were refined with restraints of N—H = 0.89 (2) Å and H⋯H = 1.52 (2) Å. For (4), H atoms of OH groups were found in a difference map and refined freely. All other H atoms in (1)–(4) were refined as riding.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989016017485/is5462sup1.cifCrystal structure: contains datablock(s) 1, 2, 3, 4, global. DOI: 10.1107/S2056989016017485/is54621sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989016017485/is54622sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S2056989016017485/is54623sup4.hklStructure factors: contains datablock(s) 3. DOI: 10.1107/S2056989016017485/is54624sup5.hklStructure factors: contains datablock(s) 4. DOI: Click here for additional data file.10.1107/S2056989016017485/is54621sup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016017485/is54622sup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016017485/is54623sup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016017485/is54624sup9.cmlSupporting information file. DOI: 1513710, 1513709, 1513708, 1513707CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Two 2-[sulfanyl]acetamide derivatives have folded conformations with the pyrimidine ring being inclined to the benzene ring by 58.64 (8) and 78.33 (9)°. 14H17N5OS (I) and C13H15N5O2S (II), the dihedral angle between the pyrimidine and benzene rings is 58.64 (8)° in (I) and 78.33 (9)° in (II). In both compounds, there is an intramolecular C—H⋯O hydrogen bond, and in (II) there is also an intramolecular N—H⋯N hydrogen bond present. In the crystals of both compounds, a pair of N—H⋯N hydrogen bonds links the individual molecules to form inversion dimers with R22(8) ring motifs. In (I), the dimers are linked by N—H⋯O and C—H⋯O hydrogen bonds, enclosing R21(14), R21(11) and R21(7) ring motifs, forming layers parallel to the (100) plane. There is also an N—H⋯π interaction present within the layer. In (II), the inversion dimers are linked by N—H⋯O hydrogen bonds enclosing an R44(18) ring motif. The presence of N—H⋯O and C—H⋯O hydrogen bonds generate an R21(6) ring motif. The combination of these various hydrogen bonds results in the formation of layers parallel to the (1-11) plane.In the title compounds, C One such important property is its inhibition potency against cancer targets. As a result of the limited capacity of drugs that can cure or at least prolong the survival of cancer patients, there is always an strong requirement for new chemotherapeutics. It has been reported that diaminopyrimidines show inhibition against cyclin-dependent kinases (cdks), thus arresting cell proliferation in cancer cells (Mesguiche P21/c and compound (II)PN-(2-methylphenyl)acetamide, viz. 1.763 and 1.805 Å, respectively  ring motif, and in compound (II)S(7) ring motif (see Tables 1The molecular structures of compounds (I)ii hydrogen bonds form a chain running along the c-axis direction. The N2—H2A⋯O1ii and C13—H13C⋯O1ii hydrogen bonds generate an A⋯O1ii and N5—H5⋯O1ii hydrogen bonds form an ii and C13—H13⋯O1ii hydrogen bonds generate an B⋯π interaction present within the layer  Å. The N1—H1A⋯N3i hydrogen bond generates an inversion dimer with an bc plane are formed.The hydrogen-bonding geometry of compounds (I)f Table 1. There ir Table 1, with thf Table 1. As a reB⋯O2iii hydrogen bond forms an iii generates an A⋯N3i hydrogen bonds, forming an inversion dimer with an B⋯O1ii and N1—H1A⋯N3i generate an In compound (II)f Table 2. These tf Table 2. The comet al., 2016N-phenylacetamide yielded seven hits. Three of these involve  groups. They include the 2-methylphenyl analogue, 2-[sulfanyl]-N-(2-methylphenyl)acetamide -2-[ sulfanyl]acetamide acetamide : To a solution of 4,6-diamino-pyrimidine-2-thiol  in 25 ml of ethanol in a round-bottom flask, potassium hydroxide  was added and the mixture was refluxed for 30 min. 2,4-Dimethylphenyl acetamide (3.52 mmol) was added and the mixture was refluxed for 3 h. At the end of the reaction (observed by TLC), the ethanol was evaporated under vacuum and cold water was added. The precipitate formed was filtered and dried to give compound (I)Compound (II): To a solution of 4,6-diamino-pyrimidine-2-thiol  in 25 ml of ethanol in a round-bottom flask was added potassium hydroxide  and the mixture was refluxed for 30 min. 3-Methoxyphenyl acetamide (3.52 mmol) was added and the mixture was refluxed for 3 h. At the end of the reaction (observed by TLC), the ethanol was evaporated under vacuum and cold water was added, and the precipitate formed was filtered and dried to give compound (II)Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017008143/su5373sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989017008143/su5373Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017008143/su5373IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989017008143/su5373Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017008143/su5373IIsup5.cmlSupporting information file. DOI: 1553497, 1553496CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/s41598-017-03883-7, published online 20 June 2017Correction to: This Article contains errors in Figure 3. In Figure 3A the y-axis ‘As (µg/L)’ is incorrectly given as ‘% As recovery’.In addition, Figure 3B was omitted.Finally, the Figure legend,“Figure 2 Inset Mean before ingestion”should read“B) Mean before ingestion”The correct Figure 3 appears below as Figure