The  d-fructose-2-aminoisobutyric acid (FruAib), C10H19NO7, (I), crystallizes exclusively in the β-pyranose form, with two conformationally non-equivalent molecules [(IA) and (IB)] in the asymmetric unit. In solution, FruAib establishes an equilibrium, with 75.6% of the population consisting of β-pyranose, 10.4% β-furanose, 10.1% α-furanose, 3.0% α-pyranose and <0.7% the acyclic forms. The carbohydrate ring in (I) has the normal 2C5 chair conformation and the amino acid portion is in the zwitterion form. Bond lengths and valence angles compare well with the average values from related pyranose structures. All carboxyl, hydroxy and ammonium groups are involved in hydrogen bonding and form a three-dimensional network of infinite chains that are connected through homodromic rings and short chains. Intramolecular hydrogen bonds bridge the amino acid and sugar portions in both molecules. A comparative Hirshfeld surfaces analysis of FruAib and four other sugar–amino acids suggests an increasing role of intramolecular heteroatom interactions in crystal structures with an increasing proportion of C—H bonds.The title compound, alternatively called  The molecular structures and atomic numbering are shown in Figs. 2d-fructose, and an amino acid, 2-aminoisobutyric acid, which are joined through the common amino group. The β-d-fructopyranose rings of the carbohydrate portions in both (IA) and (IB) exist in the 2C5 chair conformation, with puckering parameters Q = 0.582 Å, q = 177.7°, and f = 224° for (IA) and Q =0.565 Å, q = 175.5°, and f = 268° for (IB). These parameters correspond to a conformation with the lowest energy possible for fructose  providing a better fit. The bond distances and the valence angles are close to the average values for a number of crystalline pyranose structures , the vicinal proton–proton coupling constants J3,4 = 9.8 Hz and J4,5 = 3.4 Hz indicate H4 is in the trans disposition to H3 and in the gauche disposition to H5. Hence, the predominant conformation of FruAib in solution is the 2C5 β-d-fructopyranose, as well.Crystalline FruAib has two conformationally nonequivalent molecules, (IA) and (IB) are in the zwitterion form with a positively charged tetrahedral secondary ammonium nitrogen and a negatively charged deprotonated carboxyl group. Each molecule has three intramolecular interactions (Table 1B), the string of short heteroatom contacts stretches from O4B through O7B and can be denoted in terms of the A), the intramolecular hydrogen bonding is fragmented between the shorter zwitterionic bridge O7A⋯H1NA⋯O6A . Thus, hydrogen bonds form a three-dimensional network of short heteroatomic contacts throughout the crystal of (I)A) provides most of donors for these contacts.FruAib crystallizes in the triclinic space group is Fig. 4. The baset al., 2016d-fructose-2-aminoisobutyric acid: thus the compound appears to be novel. The d-fructosamine portion of the molecule is more interesting for a structure comparison survey due to its conformational instability and practical significance to food and health sciences. The most closely related structures are d-fructose-glycine . On the other hand, (IB) is structurally close to both FruGly , (IB) and structurally close sugar-amino acids. There is an obvious trend towards decrease in the proportion of intermolecular O⋯H contacts as the number of the C—H bonds in the structure increases, although a total number of hydrogen-bonds per molecule increases as well.A compendium of structures close to (I)d-glucose , and sodium acetate  were dissolved in 100 ml of a methanol/glycerol (3:1) mixture and refluxed for 3 h. The reaction progress was monitored by TLC on silica. The reaction mixture was diluted with 900 ml of water and passed through a column charged with 80 ml of Amberlite IRN-77 (H+-form). The target compound was then eluted with 0.2 M pyridine, and fractions containing pure FruAib were pooled and evaporated. The residue was redissolved in 100 ml of water, decolorized with 0.5 g of charcoal and evaporated to a syrup. The latter was dissolved in 30 ml of ethanol and made nearly cloudy with dropwise addition of acetone. Crystallization occurred within a week at room temperature. Yield 2.0 g . Major (β-pyranose anomer) peaks (ppm) in the 13C NMR spectrum in D2O: 179.35 (C8); 98.33 (C2); 72.39 (C4); 72.21 (C3); 71.79 (C5); 67.00 (C7); 66.68 (C6); 51.72 (C1); 24.66, 24.47 . See Supporting Table S1 for minor peak assignments in the spectrum. Major signals (ppm) and resolved coupling constants (Hz) in the 1H NMR spectrum: 4.038 ; 4.021 ; 3.903 ; 3.784 ; 3.775 ; 3.297 ; 3.210 ; 1.517 ; 1.512 ; J1A,1B = −12.7; J3,4 = 9.8; J4,5 = 3.4; J5,6A = 1.3; J6A,6B = −12.9.2-Aminoisobutyric acid , Uiso(H) = 1.2Ueq(methine or methylene) or 1.5Ueq(methyl). As a result of the unrealistic value obtained for the Flack absolute structure parameter [−0.5 (3) for 2254 quotients  was assigned on the basis of the known configuration for the starting compound d-glucose  I. DOI: 10.1107/S2056989017018060/zs2396Isup2.hklStructure factors: contains datablock(s) I. DOI: 1583254CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II-dicyanamide network composed of μ1,5-dicyanamide-bridged CoII chains inter-connected via μ1,5-dicyanamide bridges.The title structure comprises a cation-templated anionic Co 2H5)4]2[Co2(C2N3)5(H2O)4](C2N3), is a new example of a metal–dicyanamide coordination polymer which exhibits a unique three-dimensional framework of covalently linked CoII chains. All bridging dicyanamide ligands in the title structure are in the μ1,5-bridging mode. The anionic CoII-dicyanamide network is templated by tetraethylammonium cations residing in a series of channels extending along the b axis where additional non-coordinating dicyanamidate anions are also located. The framework structure is further stabilized by O—H⋯N hydrogen bonding between aqua ligands and dicyanamido ligands or the dicyanamide anion. In addition, C—H⋯N interactions are present between the tetraethylammonium cations and dicyanamide amide nitrogen atoms.The structure of the title compound, [N(C MII(dca)2 (dca = dicyanamide), complexes derived from MII(dca)2 by including a co-ligand, and cation-templated anionic [MII(dca)n]n) networks 2 to networks of reduced dimensions when a co-ligand or a counter-cation is included. Much of the interest in metal–dicyanamide coordination polymers has been focused on their structural diversities and their magnetic properties, particularly the long-range ferromagnetic ordering observed in some of the MII(dca)2 networks 2 complexes, there are fewer examples of cation-templated anionic [MII(dca)n]n) networks. We recently prepared the title compound, (N(C2H5)4)2[Co2(H2O)4(C2N3)5](C2N3), as a new example of a cation-templated metal–dicyanamide coordination polymer. The title structure presents a unique single three-dimensional network of covalently linked chains rather than a two-dimensional structure as commonly observed in many other metal–dicyanamide coordination polymers.Dicyanamide is a versatile ligand in the design and synthesis of coordination polymers due to its ability to coordinate to transition metal ions in a number of different modes involving some or all of its three nitrogen atoms  x, 2 − y, −z; (xi) −x, y, −z] of two symmetry-generated dicyanamide ligands. The asymmetric unit also contains two tetraethylammonium counter-ions and a non-coordinating dicyanamide anion. Two nitrogen atoms, N3/N3′ of one terminal ligand and N55/N56 of the anion, are disordered and were refined over two sets of sites.In the asymmetric unit of the title coordination polymer, there are two Cond Fig. 1. Co2 is μ1,5-dca-bridged corrugated CoII chain can be seen parallel to the [101] direction and is composed of CoII ions generated by a c glide plane parallel to the ac plane. Among the four dca ligands on each CoII cation, three are in μ1,5-bridging mode with two bridging within the same chain and one bridging to another chain. The remaining fourth dca ligand  is mono-dentate non-bridging. In the chain, the distances between two neighboring CoII atoms linked by μ1,5-dca ligands alternate between 8.1484 (8) Å (Co1⋯Co2) and 8.5620 (8) Å . All of the inter-chain Co⋯Co distances across μ1,5-dca bridges are of the same length, viz. 8.5517 (8) Å. These distances are similar to other single μ1,5-dca bridges reported in the literature  x, 2 − y, z; Co1ix⋯Co2v; symmetry code: (ix) x, y, z, and Co2ix⋯Co1x; symmetry code: (x) x, −y, 1 + z], resulting in a single three-dimensional network of covalently linked parallel chains. This is in contrast to the layered structures observed in a number of [MII(dca)n]n) networks that exhibit parallel sheets linked in the third dimension via μ1,5-dca ligands n]n) networks, interpenetration is not observed in the title structure due to the presence of tetraethylammonium cations in the void space, making these structures potential candidates for investigating their ability of storing guest molecules.In the crystal, a ns Fig. 2 and dca−MII(dca)2 networks. Upon introducing co-ligands or counter-ions, the derived MII(dca)2nL (L: co-ligand) and cation-templated [MII(dca)n]n) complexes display hydrogen bonding. In most of the MII(dca)2 derivatives, the hydrogen bonds are of non-classical C—H⋯X  types  and by hydrogen bonds involving N1 and N25 of the mono-dentate non-bridging dca ligand. In addition to the O—H⋯N hydrogen bonds, C—H⋯N hydrogen bonds are also present in the title structure between C—H groups of the tetraethylammonium cations and dicyanamide amide nitrogen atoms 2·6H2O , NaN(CN)2 , and (C2H5)4NCl  were dissolved in 40 ml of deionized water to produce a dark-red solution. Upon standing for one month, irregularly shaped red crystals  suitable for X-ray diffraction were collected by vacuum filtration and washed with deionized water. Selected IR bands : 3370 (O—H), 2977 (C—H), 2300, 2273, 2255, 2236, 2182, 2141 (C≡N), 1365 (C—N amide), 1172 (C—N amine). Elemental analysis calculated for C28H48Co2N20O4: C 39.72, H 5.71, N 33.08%. Found: C 39.81, H 5.37, N 32.74%.The title compound was prepared in a reaction where Co(NOUiso(H) = 1.2/1.5Ueq(C) whereby methyl H atoms were allowed to rotate around the corresponding C—C bond. Two nitrogen atoms, N3/N3′ and N55/N56, were disordered and refined in two parts each with their respective site-occupation factors refined dependently  and with independent Ueq parameters for each of the N atoms. All of the water H atoms were obtained from a difference Fourier map and refined freely.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016016637/wm5330sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016016637/wm5330Isup2.hklStructure factors: contains datablock(s) I. DOI: 1510337CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The two title compounds are (diaminopyrimidin-2-yl)thioacetamide derivatives. In the first structure, the pyrimidine ring is inclined to the naphthalene ring system by 55.5 (1)°, while in the second, the pyrimidine ring is inclined to the benzene ring by 58.93 (8)°. In the crystals of both compounds, molecules are linked by pairs of N—H⋯N hydrogen bonds, forming inversion dimers with  16H15N5OS, (I), and C12H12FN5OS, (II), are [(diaminopyrimidine)sulfanyl]acetamide derivatives. In (I), the pyrimidine ring is inclined to the naphthalene ring system by 55.5 (1)°, while in (II), the pyrimidine ring is inclined to the benzene ring by 58.93 (8)°. In (II), there is an intramolecular N—H⋯N hydrogen bond and a short C—H⋯O contact. In the crystals of (I) and (II), molecules are linked by pairs of N—H⋯N hydrogen bonds, forming inversion dimers with R22(8) ring motifs. In the crystal of (I), the dimers are linked by bifurcated N—H⋯ and C—H⋯O hydrogen bonds, forming layers parallel to (100). In the crystal of (II), the dimers are linked by N—H⋯O hydrogen bonds, also forming layers parallel to (100). The layers are linked by C—H⋯F hydrogen bonds, forming a three-dimensional architecture.The title compounds, C Several classes of diaminopyrimidines have been reported as new therapeutic agents. Derivatives of diaminopyrimidines also exhibit anti-cancer activity (Xu The molecular structure of compound (I)The molecular structure of compound (II)A⋯N3i hydrogen bonds, forming inversion dimers with an bc plane f Table 1. The dime Table 1.via pairs of N5—H5A⋯N2i hydrogen bonds s Table 2. This tie Table 2. The laye Table 2.et al., 2016N-phenylacetamide yielded only five hits. They include two 4,6-dimethylpyrimidine analogues viz. 2--N-phenyl acetamide -2-acetamide sulfanyl]-N-2-methylphenyl)acetamide acetamide acetamide  To a solution of 4,6-diamino-pyrimidine-2-thiol  in 25 ml of ethanol, potassium hydroxide  was added and the mixture refluxed for 30 min. Then 3.52 mmol of 2-chloro-N-acetamide was added and the mixture refluxed for 2.5 h. On completion of the reaction (monitored by TLC), the ethanol was evaporated in vacuo and cold water was added. The precipitate that 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, potassium hydroxide  was added and the mixture refluxed for 30 min. Then 3.52 mmol of 2-chloro-N-(4-fluorophenyl)acetamide was added and the mixture refluxed for 4 h. On completion of the reaction (monitored by TLC), ethanol was evaporated in vacuo and cold water was added and the precipitate formed was filtered and dried to give compound (II)3OH for compound (I)4H8O2 for compound (II)Colourless block-like crystals were obtained by slow evaporation of a solution in CHUiso(H) = 1.2Ueq.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017001293/su5347sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989017001293/su5347Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017001293/su5347IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989017001293/su5347Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017001293/su5347IIsup5.cmlSupporting information file. DOI: 1529608, 1529607CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Toxoplasma gondii can invade and replicate in all nucleated cells in a wide range of host species, and infection induces IL-1β production. IL-1β plays central roles in the stimulation of the innate immune system and inflammation. However, little is known of the innate immune responses in human fetal small intestinal epithelial cells (FHs 74 Int cells) after T. gondii infection.T. gondii GFP-RH strain. Then, IL-1β production and its mechanisms of action were evaluated using ELISA, MTT cell viability assays, Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and gene-specific small interfering RNA (siRNA) transfection.FHs 74 Int cells were infected with the T. gondii triggered significant time- and dose-dependent IL-1β production. Although T. gondii activated NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes in FHs 74 Int cells, NLRP3 levels were consistently and significantly time-dependently increased, while the other inflammasomes were not. Transfection with siRNA targeting NLRP3, cleaved caspase-1 (Casp-1) or ASC significantly reduced T. gondii-induced IL-1β production, whereas T. gondii proliferation was markedly increased. Toxoplasma gondii infection activated P2X7 receptor (P2X7R) levels in FHs 74 Int cells in a time-dependent manner; however, transfection with siRNA targeting P2X7R significantly reduced T. gondii-induced IL-1β secretion and substantially increased T. gondii proliferation, which is mediated by decreased protein expression levels of NLRP3, cleaved Casp-1 and ASC. Collectively, NLRP3-dependent IL-1β secretion is mediated by P2X7R in small intestinal epithelial cells in response to T. gondii infection, thereby controlling parasite proliferation.Infection of FHs 74 Int cells by T. gondii proliferation in small intestinal epithelial cells. These results not only contribute to our understanding of the mucosal immune mechanisms of T. gondii infection but also offer new insight into the identification of innate resistance in the gut epithelium.This study revealed that the P2X7R/NLRP3 pathway plays important roles in IL-1β secretion and inhibition of The online version of this article (10.1186/s13071-017-2573-y) contains supplementary material, which is available to authorized users. Toxoplasma gondii is an obligate intracellular protozoan parasite that can invade and replicate in all nucleated cells. It is prevalent in humans and animals worldwide, and one-third of the world’s population is reportedly infected with T. gondii [T. gondii typically results from the ingestion of cysts in contaminated food or water. Oocysts containing highly infectious sporozoites are shed by infected felids, which is the definitive host of T. gondii. A single infected cat can shed millions of oocysts into the environment. These cysts can be taken up by intermediate hosts, which include virtually any warm-blooded animal, including humans. In these infected intermediate hosts, T. gondii establishes a chronic infection in the form of bradyzoite-containing tissue cysts [lamina propria to a variety of other organs in the body [Toxoplasma gondii invades the intestinal epithelium, where it provokes appropriate immune responses that depend on local and systemic conditions [T. gondii infection remain poorly understood.. gondii . Human eue cysts . Humans the body . Intestithe body . Toxoplanditions , 5, 6. HT. gondii-infected macrophages or monocytes [T. gondii-infected intestinal epithelial cells have not been studied.The innate immune system plays key roles in sensing pathogens and triggering biological mechanisms to control infection and eliminate pathogens , 8. It ionocytes –18. HoweThe IL-1 cytokine family consists of 11 members with different roles in inflammation. IL-1β is recognized as one of the earliest and most potent pro-inflammatory agents synthesized and released in response to infectious agents and injuries , 20. UndT. gondii infection [The purinergic P2X7 receptor (P2X7R) is a bi-functional adenosine triphosphate (ATP)-gated plasma membrane ion channel that contributes to the control of many physiological functions, including cell death, killing infectious organisms, and regulating inflammatory processes –23. P2X7nfection –27.T. gondii infection [T. gondii infection have not been presented. Thus, we investigated IL-1β production and its roles in human fetal small intestinal epithelial cells (FHs 74 Int cells) after T. gondii infection using ELISA, MTT cell viability assays, Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and gene-specific small interfering RNA (siRNA) transfection.Although many reports have described IL-1β production by macrophages and dendritic cells after nfection , 24, 25,T. gondii RH strain, which expresses transgenic green fluorescent protein (GFP-RH), were maintained as described previously [v/v) mixture of DMEM/F12 supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and an antibiotic-antimycotic solution . ARPE-19 cells were infected with T. gondii at a multiplicity of infection (MOI) of 5 and incubated at 37 °C under 5% (v/v) CO2 for 2–3 days. After spontaneous host cell rupture, parasites and cellular debris were pelleted by centrifugation and washed in cold PBS. The final pellet was resuspended and passed through a 26-gauge needle fitted with a 5.0 μm pore-sized filter .Tachyzoites of the eviously  with minv/v) FBS, an antibiotic-antimycotic solution, and 30 ng/ml human epidermal growth factor  at 37 °C in a humidified atmosphere at 5% (v/v) CO2. The medium was changed every 2–3 days.A non-transformed human fetal small intestinal epithelial cell line (FHs 74 Int cells) was purchased from ATCC and cultured in DMEM with 10% . Cytokine concentrations in the samples were calculated using standard curves based on the known quantities of recombinant cytokines.FHs 74 Int cells were mock-infected (negative control) or infected with the A 3--2,5-diphenyltetrazolium bromide (MTT) assay was performed according to the manufacturer’s instructions. FHs 74 Int cells were plated in triplicate in 96-well plates at a density of 2500 cells/well and infected at MOIs of 1, 2, 5, 10 or 20. After 8 h, 5 mg/ml MTT  in PBS was added to each well at a final concentration of 0.5 mg/ml, and the plates were incubated at 37 °C for 4 h. Subsequently, formazan was dissolved in 100 μl of crystal dissolving buffer (0.04 N HCl in absolute isopropanol) and added to each well. The optical density at 490 nm was then measured using a Multiscan GO microplate reader .T. gondii GFP-RH strain as indicated. After the cells were washed with ice-cold PBS, proteins were extracted with RIPA buffer . Protein concentrations were determined using the Bradford assay . Total protein (30 μg) was resolved on 10–12% SDS-PAGE gels and then transferred to PVDF membranes . The membranes were blocked with 5% nonfat skim milk in TBS containing 0.1% Tween 20 (TBST) for 1 h and incubated with primary antibodies against IL-1β, NLRP1, NLRP3, NLRC4, AIM2, cleaved Casp-1 (Asp297), Casp-1, ASC, P2X7 receptor (P2X7R) , or α-Tubulin  overnight at 4 °C. Subsequently, the membranes were incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology) for 2 h at room temperature. Blots were developed using a commercially available enhanced chemiluminescence (ECL) detection kit . These experiments were repeated at least three times with similar results. Quantification of band intensity was performed using ImageJ software . The results were normalized to α-Tubulin protein levels and are expressed as fold changes over the mock-infection control group.Control FHs 74 Int cells or siRNA-transfected FHs 74 Int cells were mock-infected or infected with the T. gondii at an MOI of 10 for 4 or 8 h. Total cellular RNA was extracted using TRIzol Reagent , and 3 μg RNA was reverse-transcribed in a final volume of 20 μl using Superscript II reverse transcriptase (Invitrogen Life Technologies) as described by the manufacturer. qRT-PCR was performed using SYBR Premix Ex Taq II reagent  as described previously [–ΔΔCt method.FHs 74 Int cells were mock-infected or infected with the GFP-RH strain of v/v) CO2. The knockdown efficiency was determined by Western blotting. All experiments were repeated independently three times.Scrambled negative-control siRNA and siRNAs specific for NLRP3, Casp-1, ASC and P2X7R were purchased from Santa Cruz Biotechnology. FHs 74 Int cells were transfected with the siRNAs using Lipofectamine RNAiMAX (Invitrogen Life Technologies) according to the manufacturer’s instructions. Briefly, cells were seeded in 6-well plates and grown for 24 h (70% confluence) prior to transfection. Nine microliters of Lipofectamine RNAiMAX Reagent was diluted with 150 μl of Opti-MEM and 3 μl of siRNA in 150 μl of Opti-MEMR from a 10 μM stock solution. Diluted siRNA was added to diluted Lipofectamine RNAiMAX Reagent and incubated at RT for 5 min. Finally, the siRNA-lipid complex was added to the cells, and the cells were incubated for 72 h at 37 °C in a humidified atmosphere at 5%  paraformaldehyde, and permeabilized with 0.1% (v/v) Triton X-100 in PBS (PBST) for 10 min. The coverslips were then washed with PBST and stained with Texas Red-X phalloidin (Life Technologies) to label F-actin. Finally, the coverslips were washed and mounted onto microscopy slides using a mounting medium containing DAPI (to detect nuclei) , and the cells were imaged using fluorescence microscopy. All experiments were performed on triplicate samples.To measure t-test or one-way ANOVA. A P-value < 0.05 was considered significant.All results are presented as the means ± standard deviations (SDs) of at least three independent experiments, unless otherwise indicated. Statistical significance was determined using an unpaired Student’s T. gondii, FHs 74 Int cells were infected with live T. gondii at different MOIs for 8 h, and IL-1β levels in the cell culture supernatants were measured by ELISA. As shown in Fig. T. gondii significantly enhanced IL-1β secretion in an MOI-dependent manner (MOI 1: t(4) = 4.78, P = 0.009; MOI 2: t(4) = 6.756, P = 0.0025; MOI 5: t(4) = 11.72, P = 0.0003; MOI 10: t(4) = 11.52, P = 0.0003; MOI 20: t(4) = 20.36, P < 0.0001). However, the cell viability of FHs 74 Int cells infected with T. gondii at an MOI of 20 was slightly reduced compared with that of uninfected control cells  = 2.887, P = 0.045; 2 h: t(4) = 8.039, P = 0.0013; 4 h: t(4) = 11.06, P = 0.0004; 8 h: t(4) = 26.69, P < 0.0001; 12 h: t(4) = 11.69, P = 0.0003)  = 4.067, P = 0.015; 4 h: t(4) = 10.67, P = 0.0004; 8 h: t(4) = 13.39, P = 0.0002; 12 h: t(4) = 8.333, P = 0.0011)  Fig. . SimilarT. gondii infection. According to previous reports, NLRP1, NLRP3, NLRC4 and AIM2 inflammasomes are activated in T. gondii-infected DC or macrophages [T. gondii infection using qRT-PCR and Western blotting. As shown in Fig. T. gondii-infected FHs 74 Int cells. However, NLPR1 expression was not significantly changed after infection. NLRC4 mRNA levels were significantly upregulated 4 h after T. gondii infection but decreased slightly after 8 h (4 h: t(4) = 3.998, P = 0.016). In addition, AIM2 mRNA levels were significantly decreased at 8 h post-infection (t(4) = 4.213, P = 0.014). According to the mRNA and protein expression levels of 4 inflammasomes in T. gondii-infected FHs 74 Int cells, NLRP3 expression was consistently and significantly increased with time, while the others were not (4 h: t(4) = 3.047, P = 0.038; 8 h: t(4) = 4.509, P = 0.011). Therefore, we focused on the NLRP3 inflammasome. We next evaluated whether NLRP3 inflammasome is involved in T. gondii-induced IL-1β production and whether they exert protective effects in FHs 74 Int cells. In future studies, we plan to evaluate the roles of other inflammasomes.We next explored the involvement of specific inflammasomes in IL-1β secretion after rophages , 17, 18.T. gondii. As shown in Fig. T. gondii significantly upregulated NLRP3 (4 h: t(4) = 6.411, P = 0.003; 8 h: t(4) = 7.124, P = 0.002) and cleaved Casp-1 (4 h: t(4) = 8.112, P = 0.0013; 8 h: t(4) = 13.71, P = 0.0002) and ASC (4 h: t(4) = 5.494, P = 0.005; 8 h: t(4) = 8.471, P = 0.0011) protein levels in a time-dependent manner (from 2 to 8 h). To determine whether T. gondii mediates IL-1β release via the NLRP3 inflammasome, we transfected cells with siRNAs specific to NLRP3, Casp-1 or ASC for 72 h and then infected the cells with T. gondii GFP-RH at an MOI of 10 for 8 h. Cell extracts were subjected to Western blotting to detect the knockdown efficiencies of NLRP3, Casp-1 and ASC. As shown in Fig. T. gondii-induced IL-1β secretion compared with secretion in T. gondii-infected control siRNA cells (t(4) = 6.219, P = 0.003). Similarly, Casp-1 (t(4) = 4.649, P = 0.0097) and ASC (t(4) = 3.193, P = 0.033) siRNAs also significantly reduced T. gondii-induced IL-1β secretion, although IL-1β levels were not drastically attenuated  and DAPI (blue) to identify F-actin and nuclei, respectively. The number of T. gondii-infected cells and the total number of cells were counted under a fluorescence microscope, and the T. gondii infection rate was calculated. There was no significant difference in T. gondii infection rate in cells transfected with NLRP3, Casp-1 or ASC siRNA compared with those in control siRNA-transfected cells  = 20.92, P < 0.0001; Casp-1: t(4) = 7.525, P = 0.0017; ASC: t(4) = 4.504, P = 0.011)  Fig. . Taken tT. gondii. A recent study reported that P2X7R, a powerful activator of the NLRP3 inflammasome, is responsible for both NLRP3 recruitment and activation [T. gondii-induced P2X7R is involved in NLRP3 inflammasome activation, we transfected cells with P2X7R siRNA prior to T. gondii infection. Western blotting results showed that transfection with P2X7R siRNA significantly reduced T. gondii-induced NLRP3, cleaved Casp-1 and ASC protein levels  = 4.07, P = 0.0152)  = 4.421, P = 0.0115)  Fig. . To eval15) Fig. . Thus, aToxoplasma gondii can invade and replicate in the epithelial lining of the gastrointestinal tract and induce an immune response. IL-1β is a crucial factor of host defense in response to infections and injuries. Many studies have reported that T. gondii infection produces a significant amount of IL-1β in DCs, macrophages or monocytes but not in the intestinal epithelial lining [T. gondii-infected small intestinal epithelial cells, which is subsequently regulated by P2X7R and inhibits T. gondii proliferation Additional file 2: Figure S1.T. gondii at higher infectious doses (MOI 20) reduced cell viability. FHs 74 Int cells were infected with the T. gondii GFP-RH strain at the indicated MOIs for 8 h, and the viability of FHs 74 Int cells were evaluated by an MTT assay. Figure S2.Toxoplasma gondii-induced IL-1β production in FHs 74 Int cells. FHs 74 Int cells were infected with T. gondii at an MOI of 10 for different time periods, and pro-IL-1β protein levels in the cell extracts were determined by Western blotting. a Representative blots presenting the protein levels of pro-IL-1β in mock-infected or T. gondii-infected FHs 74 Int cells . b Bar plot depicting the pro-IL-1β/α-Tubulin ratio as determined by densitometric analysis of Western blotting and expressed as fold change compared with the mock-infection control. For all panels, data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the uninfected control. All data are representative of three independent experiments. (ZIP 1710 kb)FHs 74 Int cells infected with


The correct author name in the byline should read: Mátyás Gorjánácz. The correct citation should be: Santarella-Mellwig R, Franke J, Jaedicke A, Gorjánácz M, Bauer U, Budd A, et al. (2010) The Compartmentalized Bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae Superphylum Have Membrane Coat-Like Proteins. PLoS Biol 8(1): e1000281.


Numerous studies suggested that PM2.5 exposure was associated with increased risk of Alzheimer’s disease (AD). But the precise mechanisms by which PM2.5 contributed to AD pathogenesis have not been clarified.In the presence or absence of neurons, oligomeric amyloid beta (oAβ)-primed microglia were stimulated with PM2.5. Firstly, we determined the effects of PM2.5 exposure on neuronal injury and inflammation in neurons-microglia co-cultures. Then, we examined whether NLRP3 inflammasome activation was involved in PM2.5-induced inflammation. After that, we investigated whether PM2.5 exposure increased ROS level in oAβ-stimulated microglia. At last, we examined whether ROS and NLRP3 inflammasome activation was required for PM2.5-induced neuronal injury in neurons-microglia co-cultures.In the present study, we showed that PM2.5 exposure aggravated oAβ-induced neuronal injury and inflammation in neurons-microglia co-cultures via increasing IL-1β production. Further, PM2.5-induced IL-1β production in oAβ-stimulated microglia was possibly dependent on NLRP3 inflammasome activation. Meanwhile, PM2.5 exposure increased ROS level in oAβ-stimulated microglia. ROS was required for PM2.5-induced IL-1β production and NLRP3 inflammasome activation in oAβ-stimulated microglia. More importantly, ROS and NLRP3 inflammasome activation was required for PM2.5-induced neuronal injury in neurons-microglia co-cultures.For the first time, these results suggested that the effects of PM2.5 under AD context were possibly mediated by NLRP3 inflammasome activation, which was triggered by ROS. Taken together, these findings have deepened our understanding on the role of PM2.5 in AD pathogenesis. Alzheimer’s disease (AD) is the most common cause of dementia in the elderly. Unfortunately, the pathogenesis of AD still remains elusive . There iOf these environmental toxicants, PM poses severe health threats. PM is divided into three major size categories: ultra-fine PM , fine PM , and coarse PM . PM2.5 are mainly composed of compounds of both organic and inorganic, including sulfates, nitrates, carbon, ammonium, hydrogen ions, lipopolysaccharides (LPS), metals, and water . A populNeuroinflammation associated with microglia has been identified as a major contributor to AD pathogenesis . Long-teThe NLRP3 inflammasome is a cytoplasmic multiprotein complex that regulates the cleavage of IL-1β precursors. Activation of the NLRP3 inflammasome requires two signals. The first signal leads to the synthesis of pro-IL-1β and other components of the inflammasome, such as NLRP3. The second signal results in the assembly of the NLRP3 inflammasome, caspase-1 activation, and IL-1β secretion . NLRP3 iOn consideration of the above evidence, we hypothesized that PM2.5 exposure aggravated oligomeric Aβ (oAβ)-induced neuronal injury and inflammation in neurons-microglia co-cultures via increasing IL-1β production, which was mediated by NLRP3 inflammasome activation. For the first time, we show that PM2.5 exposure aggravates oAβ-induced neuronal injury and inflammation in an in vitro model of AD. Meanwhile, we reveal that the aforementioned effects of PM2.5 are mediated by NLRP3 inflammasome activation. Taken together, these findings have deepened our understanding on the role of PM2.5 in AD pathogenesis.N-acetyl-l-cysteine (NAC) were purchased from Sigma-Aldrich . Z-VAD-FMK (pan-caspase inhibitor) and Z-YVAD-FMK (caspase-1 inhibitor) were purchased from Calbiochem . IL-1 receptor antagonist (IL-1ra) was purchased from R&D . MitoQ was purchased from Focus Biomolecules .PM2.5 was purchased from the National Institute for Standards and Technology . Aβ1-42, LPS, NADPH, lucigenin, diphenylene iodonium , and Pregnant C57BL/6 mice were provided by Animal Experiment Center of Nanjing First Hospital, Nanjing Medical University. All animal procedures were carried out in accordance with Chinese Association for Laboratory Animal Sciences Guide for Care and Use of Laboratory Animals and were approved by the Nanjing Medical University Experimental Animal Care and Use Committee.d-lysine-coated 75-cm2 flasks to form a confluent glial monolayer. Half of the medium was replaced with fresh cell culture medium every 3 days. To collect microglial cells, the cultures were shaken on a rotary shaker  at 250 rpm for 3 h. The detached microglial cells were collected by centrifugation, and the enriched microglial cell suspension was plated onto poly-d-lysine-coated 6-well plates containing poly-d-lysine-coated glass coverslips. After the cells are attached, the medium was replaced with fresh culture medium. The purity of the isolated microglia was determined by immunostaining with antibodies against Iba1. In average, over 90% of cultured cells were immunostained with Iba1.Primary microglial cells were prepared from the brains of C57BL/6 at postnatal day 1 as previously described . BrieflyPrimary neurons were prepared from the cortices of mouse embryos at embryonic day 17 (E17) as previously described . BrieflyNeurons-microglia co-cultures were performed as previously described , 22. MicoAβ was prepared as previously described , 22. Bri5 cells were collected by centrifugation and resuspended in 500 μl binding buffer. Then, 5 μl annexin V and 10 μl PI were added. After incubation at room temperature for 5 min in the dark, cells were analyzed by flow cytometry. Data were analyzed using CellQuest Pro software (BD).To evaluate apoptosis, annexin V/propidium iodide (PI) analyses were performed as previously described . BrieflyThe level of intracellular ROS was measured by DCFH oxidation using a commercial detection kit , as previously reported . Briefly5) in 24-well plates were exposed to 1 ml MitoSOX reagent working solution and then incubated for 10 min at 37 °C. Cells were collected and washed twice by buffer, and cells were analyzed by flow cytometry. Data were analyzed using CellQuest Pro software (BD).The level of mitochondrial ROS was measured by MitoSOX Red superoxide indicator staining , as previously reported . Briefly5 cells/cm2 and received different treatments. Then, 10-μl CCK-8 solution was added to each well. Absorbance at 450 nm was measured 2 h after incubation in the dark.The MTT assay was performed using a Cell Counting Kit  as described previously . Briefly4 cells per dish. Cells were washed three times with PBS and incubated with a cell-permeable redox-sensitive fluorescent dye , followed by washes with PBS. Live-cell confocal imaging was performed within 30 min of washes. Signal intensity per unit area was determined for three fields of view from three different samples.In vitro validation of oxidative capacity was performed as described previously . PrimaryMouse IL-1β ELISA Kits were obtained from Invitrogen . ELISA was performed according to manufacturer’s instruction.The following primary antibodies were used: mouse anti-Caspase-1 , rabbit anti-NLRP3 antibody , and mouse anti-β-actin antibody .Western blotting was performed as described elsewhere . Cells w7 cells and treated as described, were measured for caspase-1 activity according to the manufacturer’s instruction .Microglia cells, seeded at a density of 1 × 10NADPH oxidase activity was measured by lucigenin-enhanced chemiluminescence, as previously reported . MicroglP < 0.05.Statistical analysis was performed using SPSS software . All data are expressed as mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by the LSD procedure. If variances were unequal, the Games-Howell procedure was followed by ANOVA. The level of statistical significance was defined as P < 0.05; oAβ-PM2.5 dual stimulation: 50.02 vs. 6.77%, P < 0.05; Fig. P < 0.05; Fig. P < 0.05) and 45.14% (P < 0.05)  for 3 h (LPS priming). Inserts containing microglia were washed with fresh serum-free DMEM and then plated in the upper chamber. Then, neurons-microglia co-cultures were treated with or without oAβ (5 μM). After 12 h, neurons-microglia co-cultures were treated with or without PM2.5 (50 μg/ml) for 4 h. We measured neuronal apoptosis using flow cytometry with annexin V/PI staining. When compared with neurons in co-cultures with LPS-priming microglia, oAβ stimulation and oAβ-PM2.5 dual stimulation increased neuronal apoptosis  Fig. . When co05) Fig. .Fig. 1PMThen, we assessed whether PM2.5 induced inflammation in oAβ-stimulated microglia. Microglia were activated with LPS (1 μg/ml) for 3 h (LPS priming), and then oAβ (5 μM) was added into the culture medium. After 12 h, the cells were stimulated with PM2.5 (50 μg/ml) for 4 h. The concentration of IL-1β in the culture supernatant was then measured by ELISA. As shown in Fig. P < 0.05)  and 4.07-fold (P < 0.05), respectively   Fig. , e. We d05) Fig. , e. To fely Fig. . When co05) Fig. . Taken tP < 0.05, Figs. P < 0.05) and 2.38-fold (P < 0.05), respectively   and 2.39-fold, respectively   and 1.96-fold, respectively  , we demonstrated that PM2.5 exposure increased staining intensity/unit area in oAβ-stimulated microglia  Fig. . In ordeely Fig. . When co05) Fig. . Meanwhiely Fig. . When co05) Fig. . Taken tP < 0.05), 30.20% (P < 0.05), and 52.59% (P < 0.05), respectively , 31.42% (P < 0.05), and 45.04% (P < 0.05), respectively  were used to investigate the role of ROS in IL-1β production and NLRP3 inflammasome activation. We found that all the ROS inhibitors decreased IL-1β production and caspase-1 activity in PM2.5-oAβ-stimulated microglia. Meanwhile, we investigated the effects of ROS inhibitors and caspase-1 inhibitors on neuronal injury in neurons-microglia co-cultures. We found that ROS inhibitors and caspase-1 inhibitors reduced PM2.5-induced neuronal injury. The results indicated that ROS played a key role in the PM2.5-induced IL-1β production and NLRP3 inflammasome activation. Furthermore, ROS and NLRP3 inflammasome activation was required for PM2.5-induced neuronal injury in neurons-microglia co-cultures.Taken together, in this study, we have shown that PM2.5 exposure aggravated oAβ-induced inflammation and neuronal injury in neurons-microglia co-cultures via increasing IL-1β production. Next, we demonstrated that PM2.5-induced IL-1β production in oAβ-stimulated microglia was possibly dependent on NLRP3 inflammasome activation. Using ROS inhibitors, we demonstrated that ROS was required for PM2.5-induced IL-1β production and NLRP3 inflammasome activation in oAβ-stimulated microglia. Lastly, we demonstrated that ROS and NLRP3 inflammasome activation was required for PM2.5-induced neuronal injury in neurons-microglia co-cultures. These data provide in vitro evidence that PM2.5 exposure increased neuronal injury and inflammation in microglia under AD context. Importantly, we showed that inhibition of NLRP3 inflammasome prevented PM2.5-induced neuronal injury and inflammation, suggesting that it might be a useful therapeutic target in alleviating the harmful health effects of PM2.5 exposure. Meanwhile, the findings should be further confirmed in animal models of AD.


The structure of the organic cage imposes a conduction pathway that is necessarily three-dimensional. The cage molecules also promote proton transfer by confining the water molecules while being sufficiently flexible to allow hydrogen bond reorganization. The proton conduction is explained at the molecular level through a combination of proton conductivity measurements, crystallography, molecular simulations and quasi-elastic neutron scattering. These results provide a starting point for high-temperature, anhydrous proton conductors through inclusion of guests other than water in the cage pores.Proton conduction is a fundamental process in biology and in devices such as proton exchange membrane fuel cells. To maximize proton conduction, three-dimensional conduction pathways are preferred over one-dimensional pathways, which prevent conduction in two dimensions. Many crystalline porous solids to date show one-dimensional proton conduction. Here we report porous molecular cages with proton conductivities (up to 10 Proton conduction is a fundamental process for fuel cell development, but three-dimensional proton conduction in crystalline porous solids is rare. Here, the authors report organic molecular cages in which the structure imposes three-dimensional proton conductivity competing with metal-organic frameworks. Proton exchange membrane fuel cells (PEMFCs) are an important clean energy platform. The performance-limiting component in PEMFCs is often the proton exchange membrane (PEM), which facilitates fast and selective proton transport14568912141517et al.−2 S cm−1 along the 1D channel axis but only 5.0 × 10−6 S cm−1 perpendicular to this axis (98 % relative humidity (RH), 298 K). Müllen et al.21−2 S cm−1  in the case of hexakis(p-phosphonatophenyl)benzene.In principle, the rational design of architecture in crystalline porous molecules allows us to tune proton conductivity and improve our understanding of proton conduction mechanisms, as relevant to both materials science and biologyOne limitation of proton conduction in MOFs is the tendency for directional proton transport, which in turn arises from the low-dimension pore structures in most frameworks tested232526282930CC3 (ref. RCC1 (ref. 12RCC1)12+·12Cl−·4(H2O)  1, , the proCC3 can reversibly adsorb up to 20.1 wt% water, which equates to approximately 12 H2O molecules per cage2O molecules can be located by single-crystal X-ray diffraction  under similar conditions (6.6 × 10−6 S cm−1)CC3 calculated from the Arrhenius plot at 98 % RH was 0.11 eV, which is lower than the cucurbituril material (0.31–0.56 eV)CC3 are beneficial for proton transport in comparison to the 1D proton transport pathways found in many MOFs.The neutral, crystalline cage solid  SC-XRD; , but the 30–95 % , with a  95 % RH . This isCC3, we investigated a series of protonated cages. Crystallization of an amine cage 12+ molecules in the asymmetric unit 12+ organic cations pack around fourfold screw axes parallel to the crystallographic c axis (2O molecules (12RCC1)12+ cage cavities was assigned as partially occupied H2O. There is no evidence of chloride anions occupying the cage cavities, which is central to the resulting proton conduction mechanism. A number of the chloride anions and H2O molecules were disordered over multiple positions and are clearly mobile in the structure, even at 100 K. PXRD data indicates that the same crystalline phase is retained after proton conductivity measurements  and the channels running in-between the cages (the extrinsic pores). The chloride ions, located just outside the cage window, form a gateway connecting these two kinds of pores. At 95% RH, molecular simulations suggest that water clusters are formed inside the cage cavities (consistent with X-ray data), while hydrogen-bonded chains of water molecules exist in the extrinsic pores  is required to advance long-range proton migration. Indeed, diffusion of a hydronium ion over a short distance in the extrinsic void was observed along the MEP shown in 1.Proton transport in the extrinsic void space in 1, tetrahedral (SO4)2− anions were introduced with a much larger radius than the spherical chloride anions (2.90 versus 1.67 Å). Crystallization of RCC1 from dilute H2SO4 (aq.) afforded salt 2. The SC-XRD structure of 2 was refined with Fdd2 symmetry as (H12RCC1)12+·6(SO4)2−·27.25(H2O) (2O molecules were located in the intrinsic cage cavity. The (SO4)2− anions and H2O form a 3D hydrogen-bonded network (12RCC1)12+ molecule  . Sulfate network , and themolecule . The watat 293 K , while i1, the crystal structure of 2 transforms on changing temperature or water content . On the other hand, the activation energy determined for 2 from the Arrhenius plot 2−) sitting at the window. However, it is considerably more difficult for this water molecule to traverse the window in 2 than in 1; the window-crossing event corresponds to the reaction coordinate varying between ca. 3.5 Å and ca. 4.5 Å 2− ions. Similarly, the diffusion of water in the extrinsic voids requires significantly larger activation in 2 than in 1 2− ions, leading to considerably restricted diffusive motions of water. This explains the higher proton conductivities observed for neutral CC3 up to 60% RH, which does not impose similar restrictions on the translational diffusion of water. The increase in conductivity with relative humidity is most significant for 2  is required to facilitate long-range proton conduction.These simulations rationalize the different proton conductivities measured for nt for 2 , where t0.10 eV, , indicatCC3, both 1 and 2 have strong ionic character, and the anions are pivotal in maintaining the crystal packing and in facilitating proton conduction. Both Cl− and (SO4)2− ions are powerful hydrogen-bond acceptors, and acidic protons and proton holes (OH−) can be generated through dissociation of H2O when these anions are hydrated. Even without the dissociation of H2O, the elongated O–H bond in the Owater–Hwater···anion hydrogen-bond complex will free up the oxygen atom of H2O to accept extra protons. Hence, the incorporation of charged ions into otherwise neutral porous cages increases the concentration of protons and/or proton carriers, thus increasing the protonic conductivity.Unlike 1 and 2 showed a near-linear temperature dependence (10–323 K), indicating that the movement of protons in the system remains essentially harmonic throughout. The hydrated samples of 1 and 2 show an increase in displacement at ∼200 K, which relates to the onset of diffusive motions; that is, rotation or translation of water molecules in the structure above this temperature. However, no significant quasi-elastic scattering was observed using HFBS between 200 and 303 K, possibly because the dynamics in these samples are too rapid for the instrument to measure .QENS can probe the dynamics of bulk water and confined water38−12–10−10 s) shows quasi-elastic scattering at temperatures above 220 K that is distinguishable from the resolution function of the instrument measured at 50 K  significantly improved the fit of spectra at T≥270, which is indicative of at least two diffusive behaviours in the system. The extracted line widths of the narrow function ) did not show pronounced Q2-dependence  was dissolved in CHCl3 (10 ml) by stirring. Hydrogen chloride  was added dropwise. White precipitate appeared and the reaction mixture was stirred for a further 2 h at room temperature. The precipitate was collected by filtration then washed by CHCl3 (3 × 20 ml). 1  was obtained as a white solid after being dried under vacuum at 90 °C. mp: decomposes > 220 °C; 1H NMR  δ 7.68 , 4.41 , 3.52 p.p.m. ; 13C NMR : δ 132.7, 132.1, 50.6, 42.8 p.p.m. HRMS (ES/APC+) calc. for RCC1, C48H72N12 [M+H]+ 817.6076, found 817.6076. Elemental analysis calcd (%) for (H12RCC1)12+·12Cl−·4H2O : C 43.45, H 6.99, N 12.67, Cl 32.07; found: C 43.10, H 6.85, N 12.47, Cl 31.90. IR  3379 (m), 2955 (m), 2737 (s), 2420 (w), 1582 (w), 1445 (s), 1180 (m), 1032 (m), 893 (m), 779 (m), 712 (m), 509 (m) cm−1.2SO4 aqueous solution  was added to RCC1  in H2O (5 ml) with stirring. White precipitate appears and the reaction mixture was stirred for a further 1 hour at room temperature. The precipitate was collected by filtration and recrystallized in H2O. 2  was obtained as a colourless block crystals. mp: decomposes >210 °C; 1H NMR  δ 7.70 , 4.35 , 3.50  ppm; 13C NMR : δ 133.5, 131.8, 51.0, 43.8 p.p.m. HRMS (ES/APC+) calc. for RCC1, C48H72N12 [M+H]+ 817.6076, found 817.6057. Elemental analysis calculated (%) for (H12RCC1)12+)·6(SO4)2−·21.5H2O: C 32.15, H 7.14, N 9.37, S 10.73; found: C 32.14, H 6.83, N 9.36, S 10.59. IR  3348 (w), 2987 (m), 2667 (w), 2453 (w), 1616 (m), 1464 (w), 1041 (s), 970 (w), 789 (w), 719 (w), 608 (s) cm−1.H1H NMR, 13C NMR spectra, TGA plots, water isotherms and scanning electron microscopy images of compounds 1 and 2 . The assembled Swagelok cell was connected to an EC Labs Biologic VMP3 potentiostat using banana plug cables. 2 probe (quasi four probe) electrochemical impedance spectroscopy was measured using a sinusoidal perturbation of 100 mV over the frequency range 100–1 MHz. To investigate the effect of humidification and temperature, a Memmertt Celsius humidity chamber was used. Impedance measurements were taken between 30–95% relative humidity and 303–383 K. For the humidity investigation, an equilibration time of 4 h was required between taking measurements in order for water sorption to stabilize.12RCC1)12+·12Cl−·4(H2O) (1) was measured at beamline I19, Diamond Light Source, Didcot, UK using silicon double crystal monochromated radiation (λ=0.6889 Å)12RCC1)12+·6(SO4)2− (2) were measured on a Rigaku MicroMax-007 HF rotating anode diffractometer . Empirical absorption corrections using equivalent reflections were performed with the program SADABSF|2 by SHELXLSingle-crystal X-ray data for (H12RCC1)12+·12Cl−·4(H2O) (1); CCDC entry 1,452,674. Formula C48H90N12Cl12O4; M=1,324.73 g mol−1; tetragonal space group P41, colourless crystal; a=20.153(6) Å, c=31.892(9) Å; V=12952(8) Å3; ρ=1.359 g cm−3; μ=0.509 mm−3; F (000)=5,584; crystal size=0.21 × 0.20 × 0.17 mm; T=100(2) K; 182,229 reflections measured (0.62<θ<24.84°), 24,589 unique (Rint=0.0613), 23,143 (I > 2σ(I)); R1=0.0728 for observed and R1=0.0774 for all reflections; wR2=0.1872 for all reflections; max/min difference electron density=1.507 and −0.401 ėÅ−3; data/restraints/parameters=24,589/85/1,469; GOF=1.091. Flack parameter 0.23(2). The structure was refined with the twin law Crystal data for (H12RCC1)12+·6(SO4)2−·27.25(H2O) (2); CCDC entry 1,452,672. Formula C48H138.50N12O51.25S6; M=1,896.56 g mol−1; orthorhombic space group Fdd2, colourless crystal; a=32.757(2) Å, b=34.249(2) Å, c=32.016(3) Å; V=34877(4) Å3; ρ=1.445 g cm−3; μ=0.263 mm−3; F (000)=16264; crystal size=0.17 × 0.13 × 0.12 mm; T=100(2) K; 117,747 reflections measured (1.999<θ<29.128°), 23450 unique (Rint=0.0600), 22,347 (I>2σ(I)); R1=0.0660 for observed and R1=0.0684 for all reflections; wR2=0.1854 for all reflections; max/min difference electron density=1.231 and −0.626 ėÅ−3; data/restraints/parameters=23,450/131/1,302; GOF=1.040. Flack parameter 0.115(14).Crystal data for , combined with the climbing-image nudged elastic band methodhttps://www.cp2k.org). All DFT calculations made use of the Becke–Lee–Yang–Parr (BLYP)4954−7 Hartree. To probe proton transfer in 1 under aqueous conditions, we first identified thermodynamically favourable adsorption sites for water with the aid of classical simulations. Based on snapshots thus generated for 95 % RH at 298.15 K, climbing-image nudged elastic band calculations were then performed to identify and characterize minimum-energy pathways connecting possible proton sites. Classical, force-field-based molecular dynamics and Monte Carlo simulations were used to study the dynamics of water in 1, 2, CC3, and bulk; computational details are presented in the Proton mobility in Kα radiation. Data were measured over the range 4–50° in ∼0.013° steps over 60 min. Laboratory PXRD data were collected from samples contained in borosilicate glass capillaries in transmission geometry on a Panalytical Empyrean diffractometer producing Cu Kα radiation and equipped with an X-ray focussing mirror. Data were collected using a PIXcel 3D detector in 1D scanning mode. For variable temperature PXRD measurements, the temperature of the capillary was controlled using an Oxford Cryosystems 700 Series Cryostream Plus. Patterns were indexed and lattice parameters extracted by Le Bail fitting in TOPAS AcademicPXRD data were collected in transmission mode on loose powder samples held on thin Mylar film in aluminium well plates on a Panalytical X'Pert PRO MPD equipped with a high throughput screening (HTS) XYZ stage, X-ray focusing mirror and PIXcel detector, using Cu 1, and 2 were placed in an aluminium foil pouch (of thickness sufficient to maintain a 10 % scatterer) and rolled in to an annulus and placed inside an aluminium cell filled with helium and sealed. Temperature was maintained inside a closed-cycle refrigerator equipped with a Lakeshore temperature controller to better than 0.2 K variation over time. QENS measurements using an instrument configured for the highest neutron flux at a wavelength of 5.0 Å, with detectors masked that contained Bragg peaks, and grouped in momentum transfer (Q) with 0.2 Å−1 bins, allows for an accessible Q range of 0.27 Å−1 to 2.27 Å−1 with an elastic energy resolution of approximately 110 meV. The Q-dependent spectra collected with a wavelength of 6 Å (The neutron scattering data was collected at the Center for Neutron Research (NCNR) of the National Institute of Standards and Technology , using HFBS and DCS. For both HFBS and DCS, samples of hydrated h of 6 Å  were fitwww.ccdc.cam.ac.uk/data_request/cif. The X-ray crystallographic data for the structures reported in this Article have been deposited at the Cambridge Crystallographic Data Centre, under deposition numbers 1452672–1452674. These data files can be obtained free of charge via How to cite this article: Liu, M. et al. Three-dimensional protonic conductivity in porous organic cage solids. Nat. Commun. 7:12750 doi: 10.1038/ncomms12750 (2016).Supplementary Figures 1-66, Supplementary Tables 1 & 2, Supplementary Note 1, Supplementary Methods and Supplementary References.Crystallographic information for 1 at 100 KCrystallographic information for 2 at 100 KCrystallographic information for 2 at 293 K


The data presented in this article are related to the research article entitled “MKP-1 negatively regulates LPS-mediated IL-1β production through p38 activation and HIF-1α expression”   Specifications TableValue of Data•This data shows an enhanced expression of HIF-1α in MKP-1 deficient macrophages •Nuclear extracts of MKP-1 deficient macrophages exhibited higher accumulation of HIF-1α along with its transcriptional co-activator p300 •Presented data has the potential to guide future exploration of the mechanisms involved in the stabilization of HIF-1α in absence of MKP-1or through increased activation of p38 MAPK.1-/- mice exhibit higher p38 and JNK activation in response to LPS . Phospho-specific antibodies against the phosphorylated form of ERK1/2, p38, JNK, as well as total ERK1/2, JNK, and β-actin were purchased from Cell Signaling Technology . Total p38 antibody was purchased from Santa Cruz Biotechnology . The IL-1β antibody was purchased from R&D Systems . The HIF-1α antibody was purchased from Bioss Inc . Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG secondary antibodies were purchased from Cell Signaling Technology, and horseradish peroxidase (HRP)-conjugated anti-goat antibody was purchased from Santa Cruz Biotechnology.2.26 cells/well the day before the experiment.Wild-type (WT), MKP-1 knockout mice were generated as previously described 2.3After the appropriate treatments, cells were washed with PBS, and harvested in RIPA buffer  containing protease inhibitor and anti-phosphatase cocktails, as previously described 2.4IL-1β, Il-6, and TNF-α cytokine levels in cell culture supernatants were measured using ELISA DuoKits (R&D Systems) as previously described 2.52, 10 mm KCl, 0.1 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 1.0 μg/ml leupeptin, and 1.0 μg/ml aprotinin) and incubated on ice for 10 min. After centrifugation at 14,000×g for 1 min at 4 °C, the supernatant (cytoplasmic) and the pellets (nuclear fraction) were collected.The cytoplasmic and nuclear fractions were separated as described previously


The binuclear molecules of the title complex show weak intermolecular C—H⋯Pd interactions, whereas the amine N—H functional groups are not involved in hydrogen bonding. N)methyl]phenyl-κC1}palladium(II)), [Pd2I2(C14H14N)2], was prepared by reaction of [Pd{C6H4(CH2NHCH2Ph)-2}(μ-OAc)]2 with NaI. It crystallizes with one discrete molecule in the asymmetric unit. The molecule presents an iodide-bridged dimeric structure with a cisoid arrangement with respect to the C,N-cyclopalladated ligands. Both PdII atoms have a slightly distorted square-planar coordination environment. Weak intermolecular contacts of the type C—H⋯Pd seem to have a significant influence on the arrangement of the molecules along the b axis in the crystal.The binuclear title complex, di-μ-iodido-bis({2-[(benzylamino-κ This is a rare example of a cyclopalladated complex containing bridging iodido ligands and one of the few C^N-cyclopalladated iodido-bridged complexes characterized by X-ray diffraction.Herein we report the synthesis and crystal structure of a iodido-bridged complex [Pd{CP21/n with one molecule in the asymmetric unit. The molecular structure can be described as a nearly planar dipalladium subunit of the type (C–N)Pd(μ-I)2Pd(C–N)  Fig. 1. Both pacisoid arrangement of the ligands observed in the title compound [Pd{C6H4(CH2NHCH2Ph)-2}(μ-I)]2, the dibromido analogue [Pd{C6H4(CH2NHCH2Ph)-2}(μ-Br)]2  whereas the other is trans to two nitrogen atoms (I2). Consequently, the Pd—I bond lengths of the I atoms trans to N [2.5959 (5) and 2.5801 (4) Å] are shorter than those of the I atoms trans to C [2.7504 (5) and 2.7030 (5) Å] because of the greater trans influence of the aryl ligands compared to that of the amino ligands. Similar values for these bond lengths and also for the C—Pd  and N—Pd  bond lengths have been found in the five structures of iodido-bridged cyclopalladated complexes reported so far (see Database survey). Selected torsion angles are collated in Table 1Owing to the One of the methylenic hydrogen atoms of the cyclopalladated dibenzylamine moiety coordinating to Pd1 participates in the formation of a non-classical intramolecular C—H⋯I hydrogen bond Fig. 1.b axis -2}(μ-Br)]2 . Such a disposition appears to be normal in dimeric halido-bridging cyclometalated complexes of d8 elements -2}(μ-I)]2.There are no hydrogen-bonding interactions involving the two NH groups. The most remarkable intermolecular interaction observed in the crystal structure is a weak hydrogen bond between the arylic hydrogen placed in position 3 of the phenylene ring attached to Pd2 (H16) and the Pd2 atom of the adjacent molecule. This interaction gives rise to the formation of a chain arrangement of molecules along the is Fig. 2. Althougis Fig. 2, it seemet al., 2016N,N-dimethylbenzylamine , 2.724 (4)– 2.694 (5) Å (trans to C); C—Pd: 1.964 (8)–2.113 (2) Å; N—Pd, 2.008 (8)–2.065 (5) Å].A search in the Cambridge Structural Database (Groom 6H4(CH2NHCH2Ph)-2}(μ-OAc)]2 , the solvent was partially removed (to ca 5 ml), and n-pentane was added (25 ml) to precipitate the title complex as an orange solid, which was collected and air-dried. Single crystals of the compound suitable for X-ray analysis were obtained by slow diffusion of n-pentane into a solution of the compound in CHCl3 at room temperature. Yield 845 mg, 0.983 mmol, 89%. Analysis calculated for C28H28I2N2Pd2 (859.2): C, 39.11; H, 3.26; N, 3.26. Found: C, 38.80; H, 3.21; N, 3.21. IR : ν(N—H) = 3261, 3201. 1H NMR : d = 3.83–3.95 , 4.18 , 4.23–4.29 , 4.65 , 6.83–6.87 , 6.92–7.00 , 7.32–7.41 , 7.67 . 13C{1H} NMR : d = 57.4 , 59.4 , 122.6 , 124.6 , 126.4 , 128.6 , 129.1 , 129.3 , 135.6 , 138.5 , 147.8 , 150.6 .To a suspension of the complex [Pd{C2) and Uiso(H) = 1.2Ueq(C). Hydrogen atoms bound to N atoms were discernible from a difference-Fourier map and were subsequently refined with N—H distance restraints [target value 0.87 (2) Å].Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017014281/wm5411sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017014281/wm5411Isup3.hklStructure factors: contains datablock(s) I. DOI: 1577834CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The molecules are linked by hydrogen bonds and an S⋯O contact to form layers parallel to the bc plane.In the title compound, the toluenesulfonamide ring and the combined ring system involving the pyridone and benzothiazole rings subtend an interplanar angle of 39.86 (4)°. The pyridone and benzothiazyl rings are linked by an intramolecular N—H 21H17N5O3S3·C3H7NO, the toluenesulfonamide ring and the combined ring system involving the pyridone and benzothiazole rings subtend an interplanar angle of 39.86 (4)°. The pyridone and benzothiazyl rings are linked by the intramolecular hydrogen bond N—Hamine⋯Nthiazole. The DMF O atom accepts two classical hydrogen bonds. The molecules are linked by hydrogen bonds and an S⋯O contact to form layers parallel to the bc plane.In the title compound, C The reaction between 1 and 2 in KOH–DMF gives an adduct for which four possible isomeric structures were considered (structures 3–6). Spectroscopic methods did not allow us to identify the product unambiguously and therefore the X-ray crystal structure was determined, confirming the exclusive presence of structure 6 in the solid state. The formation of 6 from the reaction of 1 and 2 is assumed to proceed via initial addition of the active methylene carbon atom of 2 to the double bond of 1, followed by elimination of CH3SH and cyclization via addition of the NH group to the cyano group of benzothiazole to give the favoured, kinetically and thermodynamically controlled product 6. The 1H NMR spectra of the product revealed the presence of an amino group at δ = 8.84 p.p.m. and a pyridine methylthio group at δ = 2.45 p.p.m. in solution. Compound 6 and its derivatives showed interesting preclinical biological results and are currently being patented  3.2662 (10) Å. The hydrogen bond H01⋯O99i connects the layers in the third dimension.The oyxgen atom of the dimethylformamide accepts two classical hydrogen bonds. The clearest packing feature is the formation of layers parallel to the ne Fig. 2, in whicd Table 1, togetheThe 2-pyridone ring displays the usual features of a narrow angle at nitrogen and a wide angle at the carbonyl carbon Table 2. A databd]thiazol-2-yl)-3,3-bis(methylthio)acrylonitrile (1)  was added to a solution of N-(2-cyanoacetyl)-4-methylbenzenesulfonohydrazide (2)  in dry DMF (30 ml) containing pulverized potassium hydroxide . The reaction mixture was refluxed with stirring for 2 h (TLC monitoring). After cooling, the reaction mixture was poured into ice-cold water and neutralized with HCl. The solid product was filtered off, washed with water, and dried. It was further purified from hot ethyl acetate: petroleum ether (1:1). The precipitated solid was crystallized from DMF to give yellow crystals, m.p. = 494 K, yield 78%.2-(Benzo[−1): ν 3393, 3208 , 3072 (ArCH), 2922 (CH3), 2210 (CN), 1677 (CO), 1594 (C=N), 1350, 1170 (O=S=O); 1H NMR : δ 2.42 , 2.45 , 7.42 , 7.49 , 7.56 , 7.71 , 8.06 , 8.13 , 8.84 , 11.44 . Analysis calculated for C21H17N5O3S3 (483.59): C 52.16, H 3.54, N 14.48%; found: C 52.11; H 3.48; N 14.50%; MS m/z (%): 484 , 384 (84%), 356 (100%), 283 (60%), 117 (77%).IR  with Uiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) for all others.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017015778/hg5500sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017015778/hg5500Isup2.hklStructure factors: contains datablock(s) I. DOI: 1582798CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The acyclic conformation is a minor species in the compound’s solution.The monoclinic unit contains a rare acyclic  14H21NO6, (I), crystallizes exclusively in the acyclic keto form. In solution of (I), the acyclic tautomer represents only 10% of the population in equilibrium, with the other 90% consisting of β-pyranose, β-furanose, α-pyranose, and α-furanose cyclic forms. The carbohydrate chain in (I) has a zigzag conformation and the aromatic amine group has a transitional sp2/sp3 geometry. Bond lengths and valence angles in the carbohydrate portion compare well with the average values for related acyclic polyol structures. All of the hydroxyl groups are involved in intermolecular hydrogen bonding and form a two-dimensional network of infinite chains, which are interlinked by intramolecular hydrogen bonds and organized into R88(16) homodromic ring patterns. A comparative Hirshfeld surfaces analysis of (I) and four other 1-amino-1-deoxy-d-fructose derivatives suggests the balance of hydrophilic/hydrophobic interactions plays a role in the crystal packing, favoring the acyclic isomer.The title compound, C Remarkably, in the aqueous solution of (I)keto form is a minor constituent of the established equilibrium, at 10.3% of the total population as follows from the 13C NMR data (Table 1The molecular structure and atomic numbering for (I)a Table 1. The prea Table 1.zigzag conformation, having four out of six of its carbon atoms, C3, C4, C5, and C6, located in one plane. The conformation around the carbonyl group is also nearly flat and involves atoms N1, C1, C2, O2, C3, and O3, with the carbonyl O2 in the syn-periplanar position with respect to both N1 and O3 [respective torsion angles are 11.7 (3) and −7.0 (3)°]. This type of conformation is preferred for the β-aminocarbonyl group, due to influence of the σC—H → σC=O* and σC—H → πC=O* hyperconjugation in conditions when interaction between the nitrogen lone pair (LP) and the carbonyl π*-system is not significant 13C NMR spectrum ab plane , which may qualify as weak hydrogen bonds (Table 3Compound (I)ne Fig. 4. The cars Table 3. The Hirs Table 3 revealeds Table 3.et al., 2016N-methyl-p-methoxyphenylamino)-d-fructose returned no references; hence compound (I)d-fructose-N-methyl-p-toluidine , d-fructose-p-toluidine , d-fructose-N-ethyl-p-chloroaniline , and d-fructose-N-allylaniline . Each of these 1-amino-1-deoxy-d-fructose derivatives features an aromatic substituent at the amino group. They also display a similar to (I)keto conformations in crystalline state. Frupti , crystallize in the β-d-fructopyranose anomeric form versus crystal environments was beyond the goals of the current study. However, some initial clues can be derived from analysis of data compiled in Table 1et al., 2009keto tautomer. As pointed out in Section 2, a neighboring π-system may resonate with the amine p-electrons thus making them unavailable for σ-bonding. Next, among N-aryl derivatives of 1-amino-1-deoxy-d-fructose, only those lacking a proton bound to the tertiary amino group can crystallize in the acyclic form. Indeed, no hydrogen bonds involving N1 were detected in acyclic (I)et al., 2009d-fructose derivatives reveal at least one hydrogen-mediated intramolecular heteroatom contact between the amino nitrogen atom and an oxygen atom originating from the carbohydrate portion of the molecule, most often the anomeric O2. Thus, the inability of the amino group to form stable intramolecular hydrogen bonds with the carbohydrate portion plays a role in stabilization of the acyclic tautomer. Finally, a comparative Hirshfeld surfaces analysis  to allyl or butyl (in FruAlla and d-fructose-N-butylaniline), leads to a loss of propensity to crystallize in the acyclic form  of p-anisidine and 0.55 mL of 3-mercaptopropionic acid catalyst/antioxidant was stirred for 6 h in 12 mL of isopropanol in a screw-capped glass vial at 360 K. The reaction progress was followed by TLC. The purification step included an ion-exchange on Amberlite IRN-77 (H+), with 0.2 M NH4OH in 50% ethanol as an eluant, and was followed by flash filtration on a short silica column using 5% MeOH in CH2Cl2 as an eluant. Crystallization of the compound was aided by the addition of a small amount of acetone to the syrupy evaporation residue. The crystals were filtered off, washed with acetone and dried in vacuo over CaCl2, yield 1.6 g  of colorless prisms. Major (β-pyranose anomer) peaks (ppm) in the 13C NMR spectrum in D2O/pyridine: 154.14 (C10); 148.12 (C7); 117.86, 117.11 ; 116.96, 116.80 ; 101.56 (C2); 72.98 (C4); 71.99 ; 65.90 (C6); 63.21 (C1); 57.84 (C14); 42.62 (C13). The 13C CPMAS–TOSS spectrum of finely powdered crystalline (I)The preparation of (I)Uiso(H) = 1.2Ueq(methine or methylene) or 1.5Ueq(methyl). The Flack absolute structure parameter determined [0.3 (5) for 1149 quotients  configuration which was assigned for this chain system on the basis of the known configuration for the starting material d-glucose  I. DOI: 10.1107/S2056989018000099/qm2121Isup2.hklStructure factors: contains datablock(s) I. DOI: 1811885CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


We have previously shown that Gαi3 is elevated in human glioma, mediating Akt activation and cancer cell proliferation. Here, we imply that Gαi3 could also be important for irradiation resistance. In A172 human glioma cells, Gαi3 knockdown (by targeted shRNAs) or dominant-negative mutation significantly potentiated irradiation-induced cell apoptosis. Reversely, forced over-expression of wild-type or constitutively-active Gαi3 inhibited irradiation-induced A172 cell apoptosis. Irradiation in A172 cells induced Gαi3 translocation to cell nuclei and association with local protein DNA-dependent protein kinase (DNA-PK) catalytic subunit. This association was important for DNA damage repair. Gαi3 knockdown, depletion (using Gαi3 knockout MEFs) or dominant-negative mutation potentiated irradiation-induced DNA damages. On the other hand, expression of the constitutively-active Gαi3 in A172 cells inhibited DNA damage by irradiation. Together, these results indicate a novel function of Gαi3 in irradiation-resistance in human glioma cells. Glioma is the most common primary central nervous system (CNS) malignancy. It is a major health threat –3. Each i.e. Gab1), which mediates activation of downstream Akt signaling [G protein α inhibitory subunit (Gαi) couples with GPCRs (G-protein coupled receptors)  to inhibignaling –11.There are at least three Gαi subunits, including Gαi1, Gαi2 and Gαi3 . Our recIn order to study the potential function of Gαi3 in irradiation resistance, shRNA strategy was applied. As described previously , two disThe results above suggested that Gαi3 might be important in irradiation resistance. To further support this hypothesis, Gαi3 knockout (“KO”) mouse embryonic fibroblasts (MEFs)  were utiIt is known that irradiation kills cancer cells via inducing cell apoptosis , 14. We Based on the results above, we would speculate that Gαi3 over-expression shall cause irradiation resistance. Thus, wild-type (“WT”) Gαi3 construct  wasNext, mutation strategies were employed to potentially alter the activity of Gαi3 in A172 cells. As discussed in our previous studies , 11, theIt is known that irradiation induces DNA damages, which leads to subsequent cell apoptosis –22. DNA The results of this study suggest that Gαi3 could be a key resistance factor of irradiation in glioma cells. Gαi3 depletion significantly potentiated irradiation-induced cell apoptosis. On the other hand, forced over-expression of Gαi3 inhibited irradiation-induced A172 cell apoptosis. Meanwhile, irradiation sensitivity in A172 cells was potentiated when expressing DN-Gαi3, but was reduced after CA-Gαi3 expression. Mechanistic study further showed that Gαi3 translocation to nuclei, which was important for DNA damage repair. These results together imply that Gαi3 over-expression in human glioma cells could be a key irradiation-resistance factor.Irradiation-induced DNA damage will initiate DNA repair pathway –22. TherIt is known that DNA-PKcs is vital in the repair of damaged DNA by irradiation , 23, 24.In the current study, we discovered an unique function of Gαi3: Irradiation in A172 cells induced Gαi3 translocation to nuclei, where it formed a complex with local protein DNA-PKcs. The complexation between Gαi3 and DNA-PKcs was apparently crucial for DNA repair. Gαi3 silence, depletion or dominant-negative mutation significantly potentiated irradiation-induced DNA damages. Reversely, expression of the constitutively-active Gαi3 inhibited DNA damage by irradiation in A172 cells. Future studies will be needed to further explore the detailed mechanisms of Gαi3's function in DNA damage repair.All the antibodies of the current study were described previously , 36, 37,2 incubator.Wild-type (WT) and Gαi3 knockout (KO) mouse embryonic fibroblasts (MEFs) were described previously –11. HumaCells were irradiated with a 137Cs gamma rays source at a dose rate of 1.25 Gy/min (MDS Nordion Gammacell Irradiator).Following the applied treatment, cells were lysed using the lysis buffer described , 10, 36.The detailed protocol was described in our previous studies , 36. BriThe two lentiviral Gαi3 shRNAs (“−a/−b”) were again purchased from Genechem , with the targeted sequences 5′-TCAATCATTCTCTTCCTTA-3′ (Gαi3 shRNA-a) and 5′-CCTCAGTGATTATGACCTT-3′ (Gαi3 shRNA-b), respectively. The lentiviral shRNA was added directed to the cells for 24 hours, puromycin  was added to select the stable cells. Gαi3 knockdown was confirmed by the Western blotting assay. Same amount of lentiviral scramble shRNA  was added to the control cells.The wild-type Gαi3 (-Flag), the constitutively-active-Gαi3 , the dominant-negative Gαi3 , and the empty vector  were described previously –11. The MTT assay of cell growth, clonogenicity assay of cell growth, and trypan blue staining of cell death, as well as Histone DNA apoptosis ELISA assay, Annexin V FACS assay of cell apoptosis and TUNEL nuclei staining assay of cell apoptosis were described in detail in our previous studies , 40, 41.After irradiation, cells were trypsinized and fixed in ice-cold ethanol. Afterwards, cells were incubated with a mouse monoclonal anti-γ-H2AX antibody  for 12 hours, and then incubated with a FITC-conjugated anti-mouse secondary antibody . Cells were then subjected to FACS assay to determineγ-H2AX percentage, which indicates DNA damage intensity .p < 0.05 were considered as statistically significant.The data were presented as means ± standard deviation (SD) of one whole set of experiment. All experiments were repeated at least three times, with similar results obtained in each repeat. Statistical differences were analyzed by one-way ANOVA and multiple comparisons with the post hoc Bonferroni test (SPSS version 18.0). Values of In summary, these results indicate a pivotal function of Gαi3 in irradiation-resistance in human glioma cells. Gαi3 could be a novel oncotarget for irradiation sensitization for glioma.


S-casein (αS-CN), κ-casein (κ-CN) and β-casein (β-CN) to 50.4 ± 2.9, 33.5 ± 3.0, 4.2 ± 0.5 and 1.2 ± 0.1%, respectively. The use of MTGase in conjunction MI with led to a 3-fold increase in the rate of milk protein polymerization, compared to a sample that contained MTGase but did not undergo MI. Results of two-dimensional gel electrophoresis (2-DE) indicated that κ-CN, β-CN, a fraction of serum albumin (SA), β-LG, α-lactalbumin (α-LA), αs1-casein (αs1-CN), and αs2-casein (αs2-CN) were polymerized in the milk, following incubation with MTGase and MI at 30 °C for 1 h. Based on this result, the combined use of MTGase and MI appears to be a better way to polymerize milk proteins.We investigated the combined effects of microbial transglutaminase  and microwave irradiation (MI) on the polymerization of milk proteins at 30 °C for 3 h. The addition of MTGase caused the milk proteins to become polymerized, which resulted in the formation of components with a higher molecular-weight (>130 kDa). SDS-PAGE analysis revealed reductions in the protein content of β-lactoglobulin (β-LG), α MTGase is widely used to modify the solubility, hydration, and heat stability of proteins in food products. MTGase can be used to catalyze the acyl transfer reaction between lysine residues and glutamine residues, which causes ε-(γ-glutamyl)lysine covalent bonds with intra- and inter-molecular cross-linking to form in proteins1s1-CN, αs2-CN, β-CN, and κ-CN) and whey proteins7Milk proteins include caseins (αKcat/Km) values have also been achieved in the microwave-assisted digestion of β-LG by pronaseMI has obvious advantages in certain enzymatic reactionsIn this study, we investigated the application of MI to enhance the MTGase-induced polymerization of caseins and whey proteins. No previous research has used SDS-PAGE or 2-DE to elucidate the effects of microwave-assisted polymerization on milk molecules.S-CN, κ-CN, and β-CN  crosslinking. Smiddy, Martin, Kelly, de Kruif, and Huppertzet al.2018O-labeling and mass spectrometry to identify protein cross-linking. Their strategy, which is based on XChem-Finder, can be used to identify cross-linking in individual caseins.We first investigated the effects of MI and MTGase (7.0 units/mL) on the polymerization of milk proteins . Milk saand β-CN . In agreet al.n-caprylic acid and pentanol isomers catalyzed via lipase. Wan, Sun, Hu, and XiaMolecules with high dipole moments, such as enzymes and proteins, are particularly susceptible to the effects of microwaves22S-CN, κ-CN and β-CN were reduced to 50.4 ± 2.9, 33.5 ± 3.0, 4.2 ± 0.5 and 1.2 ± 0.1%, respectively. These results indicate that the MTGase-induced polymerization of κ-CN, β-CN, and αS-CN occurred earlier than that of β-LG. De Jong and KoppelmanS-CN was noticeably lower in samples where MTGase was used in conjunction with MI than in samples which contained MTGase but did not undergo MI , αs2-CN (No. 6–8), β-CN (No. 9–10), κ-CN (No. 11–14), β-LG (No. 15–16), and α-LA (No. 17). α-LA, SA, and β-LG are components of whey proteins, whereas κ-CN, β-CN, αs1-CN, and αs2-CN are components of caseinss1-CN (No. 4–5), αs2-CN (No. 6–8), β-CN (No. 9–10), and κ-CN (No. 11–14) as well as portions of SA (No. 1–3), β-LG (No. 15–16) and α-LA (No. 17) showed signs of depletion after incubation for 3 h in the MTGase-containing milk. This indicates that most of the casein micelles and a portion of the whey proteins were polymerized by MTGase. We also observed that the casein polymerization of αs1-CN and αs2-CN was slower than that of β-CN and κ-CN. Smiddy, Martin, Kelly, de Kruif, and Huppertzs1-CN (No. 4–5), αs2-CN (No. 6–8), β-CN (No. 9–10), and κ-CN (No. 11–14), as well as portions of SA (No. 1–3), β-LG (No. 15–16), and α-LA (No. 17) showed signs of depletion after incubation for 1 h in the MTGase-containing milk  for the thermal inactivation of MTGase at 45 °C was 6.2 × 10−5/min. In addition, the residual MTGase activity in MTGase-containing milk was 99.8 ± 0.8% when the sample was incubated in a dry bath incubator at 30 °C for 0 h. These results suggest that the components of milk can not affect the activity of MTGase. Nonetheless, the residual MTGase activity in milk decreased significantly to 25.2 ± 2.7% after 3 h of incubation. Therefore, the residual MTGase activity in milk samples incubated at 30 °C for 3 h was lower than that in buffer samples (P < 0.05). As previously mentioned, we observed that caseins and whey proteins were polymerized into higher-molecular-weight proteins by MTGase, which means that MTGase was probably entrapped within the high-molecular-weight polymers, thereby leading to a reduction in residual MTGase activity.MTGase-containing buffer  was incubated in a dry bath at 30 °C for 3 h. Residual MTGase activity in the buffer decreased slightly as incubation time increased . After 3P < 0.05). The residual MTGase activity in all milk samples incubated in the microwave reactor exceeded that of the samples incubated in the dry bath incubator. This indicates that MTGase is more stable under MI, and these findings are supported by Yu et al.MTGase-containing milk samples were also incubated in a microwave reactor at 30 °C for 3 h, which resulted in a significant decrease in residual MTGase activity to just 42.4 ± 2.5%  was purchased from Ajinomoto Co. Inc. . All experiments on the microwave-assisted polymerization of milk proteins were performed using 1 mL milk samples contained within a closed vessel, where they did not undergo any stirring or shaking. To prepare milk with/without MTGase (7.0 units/mL), we first incubated the milk samples in a dry bath incubator at 30 °C for 0, 1, 2 and 3 h, respectively. The resulting samples were then heated to 80 °C for 3 min to deactivate the MTGase. The microwave-assisted polymerization of milk proteins induced by MTGase was investigated using a microwave reactor . Milk samples with/without MTGase (7.0 units/mL) were incubated in the microwave reactor at 30 °C at a power of 30W for 0, 1, 2, or 3 h. Samples were then heated to 80 °C for 3 min to deactivate the MTGase. Experiments were conducted under one of four conditions: (1) milk without MTGase or MI; (2) milk with MTGase; (3) milk with MI; and (4) milk with MTGase and MI.Milk was first centrifuged at 5,000 × g for 20 min, and a layer of fat was subsequently removed from the top of the milk using a spatula. The resulting skimmed milk was then stored at 4 °C. A Bio-Rad protein assay kit was used in accordance with manufacturer’s instructions to determine the protein concentrations in the skimmed milk, using SA as a standard. MTGase from Milk samples were analyzed using SDS-PAGE and 2-DE, in accordance with protocol outlined by Hsieh and Pan® Discover system, power of 30 W) at 30 °C for 0, 1, 2, or 3 h. This experiment was conducted under one of four conditions: (1) MTGase-containing buffer without MI; (2) MTGase-containing buffer with MI; (3) MTGase-containing milk without MI; and (4) MTGase-containing milk with MI. The resulting samples were then analyzed using MTGase assay to assess residual MTGase activity in accordance with the method proposed by Ho, Leu, Hsieh, and JiangMTGase (7.0 units/mL) was respectively added to buffer  and milk samples. The MTGase-containing buffer and milk samples were incubated in a dry bath incubator or microwave reactor .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The reaction of silver nitrate with the ligands 2,3,5,6-tetrakis[(methylsulfanyl)methyl]pyrazine, 2,3,5,6-tetrakis[(phenylsulfanyl)methyl]pyrazine and 2,3,5,6-tetrakis[(pyridin-2-ylsulfanyl)methyl]pyrazine, three tetrakis-thioether-substituted pyrazine ligands, lead, respectively, to the formation of compounds with a metal–organic chain, a metal–organic network and a metal–organic framework structure. L1) with silver(I) nitrate led to {[Ag(C12H20N2S4)](NO3)}n, (I), catena-polypyrazine] nitrate], a compound with a metal–organic chain structure. The asymmetric unit is composed of two half ligands, located about inversion centres, with one ligand coordinating to the silver atoms in a bis-tridentate manner and the other in a bis-bidentate manner. The charge on the metal atom is compensated for by a free nitrate anion. Hence, the silver atom has a fivefold S3N2 coordination sphere. The reaction of the ligand 2,3,5,6-tetrakis[(phenylsulfanyl)methyl]pyrazine (L2) with silver(I) nitrate, led to [Ag2(NO3)2(C32H28N2S4)]n, (II), polypyrazine}disilver], a compound with a metal–organic network structure. The asymmetric unit is composed of half a ligand, located about an inversion centre, that coordinates to the silver atoms in a bis-tridentate manner. The nitrate anion coordinates to the silver atom in a bidentate/monodentate manner, bridging the silver atoms, which therefore have a sixfold S2NO3 coordination sphere. The reaction of the ligand 2,3,5,6-tetrakis[(pyridin-2-ylsulfanyl)methyl]pyrazine (L3) with silver(I) nitrate led to [Ag3(NO3)3(C28H24N6S4)]n, (III), polypyrazine}trisilver(I)], a compound with a metal–organic framework structure. The asymmetric unit is composed of half a ligand, located about an inversion centre, that coordinates to the silver atoms in a bis-tridentate manner. One pyridine N atom bridges the monomeric units, so forming a chain structure. Two nitrate O atoms also coordinate to this silver atom, hence it has a sixfold S2N2O2 coordination sphere. The chains are linked via a second silver atom, located on a twofold rotation axis, coordinated by the second pyridine N atom. A second nitrate anion, also lying about the twofold rotation axis, coordinates to this silver atom via an Ag—O bond, hence this second silver atom has a threefold N2O coordination sphere. In the crystal of (I), the nitrate anion plays an essential role in forming C—H⋯O hydrogen bonds that link the metal–organic chains to form a three-dimensional supramolecular structure. In the crystal of (II), the metal–organic networks  stack up the a-axis direction but there are no significant intermolecular interactions present between the layers. In the crystal of (III), there are a number of C—H⋯O hydrogen bonds present within the metal–organic framework. The role of the nitrate anion in the formation of the coordination polymers is also examined.The reaction of the ligand 2,3,5,6-tetrakis[(methylsulfanyl)methyl]pyrazine ( L1), 2,3,5,6-tetrakis[(phenylsulfanyl)methyl]pyrazine (L2) and 2,3,5,6-tetrakis[(pyridin-2-ylsulfanyl)methyl]pyrazine (L3), were synthesized by the reaction of 2,3,5,6-tetrakis(bromomethyl)pyrazine  with silver(I) nitrate, led to the formation of a metal–organic chain (MOC) structure, (I)3N2 coordination sphere with a highly distorted shape and a τ5 value of 0.63 methyl]pyrazine (L2) with silver(I) nitrate, led to the formation of a metal–organic network (MON) structure, (II)2NO3 coordination sphere, best described as a highly distorted octahedron methyl]pyrazine (n Table 2.L3) with silver(I) nitrate, led to the formation of a metal–organic framework (MOF) structure, (III)3 unit located about a twofold rotation axis. The organic ligand coordinates to the silver atoms (Ag1), in a bis-tridentate manner. One pyridine N atom, N2, bridges the monomeric units, so forming a chain structure along the b-axis direction. The nitrate O atoms, O11 and O13, coordinate to silver atom Ag1, hence it has a highly distorted octahedral S2N2O2 coordination sphere methyl]pyrazine (e Table 3. The chaii = 2.6010 (11) Å in (III)i is 2.267 (3) Å, while bond length Ag2—N3 is shorter at 2.208 (3) Å s Table 4, formingbc plane and stack up the a axis is Fig. 6, but theca 3.7% of the total volume of the unit cell.In the crystal of (III)et al., 2016P21/n polymorph pyrazine rk Fig. 6. Finallyrk Fig. 6. Here, iCompound (I)L1  was introduced into a 16 mm diameter glass tube and layered with MeCN (2 ml) as a buffer zone. Then a solution of AgNO3  in MeCN (5 ml) was added very gently to avoid possible mixing. The glass tube was sealed and left in the dark at room temperature for at least two weeks, whereupon yellow plate-like crystals of complex (I)−1): ν = 2985 w, 2912 w, 1406 bm, 1341 bs, 1141 w, 1115 w, 982 w, 828 w, 777 w, 701 vw, 478 vw.A solution of Compound (II)L2  in MeCN (5 ml) was added very gently to avoid possible mixing. The glass tube was sealed and left in the dark at room temperature for at least three weeks, whereupon yellow block-like crystals of complex (II)−1): ν = 3053 vw, 2962 vw, 2927 vw, 1583 w, 1480 w, 1386 bs, 1278 vs, 1133 vw, 1023 w, 850 vw, 738 s, 690 m, 495 vw, 478 vw.A solution of Compound (III)L3  was introduced into a 16 mm diameter glass tube and layered with MeCN (2 ml) as a buffer zone. Then a solution of AgNO3  in MeCN (5 ml) was added very gently to avoid possible mixing. The glass tube was sealed and left in the dark at room temperature for at least two weeks, whereupon pale-yellow needle-like crystals of complex (III)−1): ν = 3097 vw, 2899 vw, 1581 m, 1562 w, 1460 m, 1386 bs, 1305 bs, 1163 w, 1126 w, 1032 vw, 1004 vw, 825 vw, 759 m, 723 vw, 461vw.A solution of A/O12B and O13A/O13B were refined with a fixed occupancy ratio of 0.5:0.5. No absorption correction was applied for complex (II)ψ scans. For complex (III)3) was corrected for using the SQUEEZE routine in PLATON Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 610.1107/S2056989017002791/wm5369sup1.cifCrystal structure: contains datablock(s) I, II, III, Global. DOI: 10.1107/S2056989017002791/wm5369Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017002791/wm5369IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989017002791/wm5369IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 1533573, 1533572, 1533571CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: