ALOX12), we identified the integrin β4 (ITGB4).Integrins and enzymes of the eicosanoid pathway are both well-established contributors to cancer. However, this is the first report of the interdependence of the two signaling systems. In a screen for proteins that interacted with, and thereby potentially regulated, the human platelet-type 12-lipoxygenase -HETE, a known bioactive lipid metabolite that regulates numerous cancer phenotypes.The net effect of the interaction was the prevention of cell death in response to starvation. Additionally, regulation of β4-mediated, EGF-stimulated invasion was shown to be dependent on 12-LOX, and downstream Erk signaling in response to ITGB4 activation also required 12-LOX.This is the first report of an enzyme of the eicosanoid pathway being recruited to and regulated by activated β4 integrin. Integrin β4 has recently been shown to induce expansion of prostate tumor progenitors and there is a strong correlation between stage/grade of prostate cancer and 12-LOX expression. The 12-LOX enzymatic product, 12(S)-HETE, regulates angiogenesis and cell migration in many cancer types. Therefore, disruption of integrin β4-12LOX interaction could reduce the pro-inflammatory oncogenic activity of 12-LOX. This report on the consequences of 12-LOX and ITGB4 interaction sets a precedent for the linkage of integrin and eicosanoid biology through direct protein-protein association.The online version of this article (doi:10.1186/s12943-015-0382-5) contains supplementary material, which is available to authorized users. The human β4 integrin subunit was identified as a 12-LOX-interacting protein, and thus a potential 12-LOX regulator , anchora12-Lipoxygenase is associated with many of the same β4-mediated phenotypes , and als12-LOX metabolizes arachidonic acid (AA) exclusively to 12(S)-HETE . This meleft panels). Conversely β4 was immunoprecipitated with an antibody to 12-LOX . The association of 12-LOX with β4 was time-dependent beginning at 5 min post stimulation. These data suggest that the interaction between 12-LOX and β4 originally discovered in the yeast 2-hybrid model system also occurs in a cultured human tumor cell model.The A431 human epidermoid carcinoma cell line has been widely used to study 12-LOX, as it expresses enzymatically active 12-LOX protein, but not the leukocyte-type isoform . Previouupper and lower panel, respectively. lane 3-CHO transfectants producing full-length, wild-type β4 alone, or lane 4-in combination with 12-LOX). Full-length β4 was recognized by antibodies to both its extracellular (3E1 mAb) and cytoplasmic (450-11A mAb) domains. 12-LOX also co-immunoprecipitated with truncated, head-less β4 , but not with truncated, tail-less integrin β4 . While truncated, tail-less β4 (95 kDa) complexes with α6 -12-HETE standard and [14C]-AA were from NEN Research Products . ODS-Silica cartridges were from J.T. Baker Inc. . FuGENE 6 Transfection Reagent kit was from Boehringer Mannheim .Human laminin and EGF were from GIBCO BRL or Sigma Aldrich . The 12-LOX-selective inhibitor, BMD122, formerly called BHPP for N-benzyl-N-hydroxy-5-phenylpentanamide , was a gA431 and Chinese hamster ovary (CHO) cells were obtained from the American Type Culture Collection , and cultured as recommended. Transfectants were selected and cultured in media with 300 μg/ml Geneticin . Prostate cancer cell lines PC-3 12-LOX/3.1 have been described previously .6 A431 cells were grown to sub-confluence in 100 mm Petri dishes and serum-starved overnight prior to use. Cells were washed with PBS (3×) and stimulated with β4 antibody for 5, 15, 30, 60 and 90 min at a concentration of 5 μg/ml in serum-free DMEM media. For experiments where the natural α6β4 ligand was immobilized, the dishes were coated with laminin (10 μg/ml) and cells were subsequently harvested as above. Otherwise, laminin was used at 5 μg/ml in serum-free media.For treatment with β4 mAb (3E1), 5 × 106) were cultured to 80 % confluence , rinsed (2×) with PBS buffer, and washed (2×) in isotonic buffer  before suspension in homogenization buffer  containing protease inhibitors. Cells were homogenized by sonication  (Vibracell-Microtip) with intervals of 3 min. In some experiments, homogenates were initially centrifuged at 10,000 × g  and the resultant supernatant was considered cytosolic. The membrane fraction represents the pellet obtained after a one-step centrifugation of the homogenate at 100,000 × g . The 10,000- and 100,000 × g pellets, respectively, were rinsed once with homogenization buffer and resuspended in protease inhibitor-free homogenization buffer. Samples, standardized by protein concentration, were immediately used for SDS-PAGE.Cells (5×105) were seeded into six well plates and serum-starved overnight. The following day media was replaced with phenol red-free RPMI media. Cells were then stimulated with 3E1 in the presence of 10 μm AA in 1 % fatty acid-free BSA. AA untreated cells served as a control. As an additional control, AA was incubated in wells without cells to measure spontaneous oxidation of AA into 12(S)-HETE, and this value was subtracted from cell-generated 12(S)-HETE values. The detailed lipid extraction protocol has been described [12(S)-HETE was measured by liquid chromatography-mass spectrometry as previously described. Cells , and subsequently clarified . Supernatants were immunoprecipitated with 4–6 μl of antibody against human 12-LOX, anti-β1, or the anti-β4 subunit for 2 h, followed by 40 μl Sepharose 4B-conjugated protein G at 4 °C overnight. Immune complexes were washed (3×) in lysis buffer, and used for SDS-PAGE. Whole cell lysates were used for input controls.Performed as per standard techniques with horseradish peroxidase-conjugated secondary anti-IgG diluted 1:4500, and enhanced chemiluminescence (ECL)  for detection.c-myc epitope tag). The full-length cDNA encoding human 12-LOX was subcloned into the EcoRI/XbaI sites of pcDNA3.1 (Invitrogen) from pCMV-12-LOX, gifted from Dr. Colin D. Funk . Expression vectors of full-length β4 and 12-LOX used neomycin as a selection marker. As deletion constructs of β4 contained no selective marker, they were cotransfected with neomycin encoding vector, pcDNA3.1. Cells grown in 6-well plates were transfected with 3–12 μg of pcDNA3.1, pCMV-β4, pcDNA-12-LOX, pCMV-β4 tail-less or pCMV-β4 headless using the FuGENE 6 Transfection Reagent following the manufacturer’s protocol. Neomycin-resistant cells were selected in 300 μg/ml geneticin. Knockdown of gene expression by shRNA was performed using Lentiviral pGIPZ constructs targeted to unique regions of the 12-LOX gene, which were purchased from Open Biosystems : V2LHS_112083 (#1), V2LHS_112086 (#2), V2LHS_112087 (#3), V3LHS_335849 (#5), V3LHS_335846 (#6), RHS4346 (#8). A431 stable transfections were achieved with 2 μg plasmid DNA, using Lipofectamine LTX (Invitrogen), followed 48 h later by selection in DMEM containing 1 ug/mL puromycin for 3 weeks . Authorization to use lenti-based vectors for transfection was granted by the WSU Institutional Biosafety Committee as protocol IBC 02-51-11.Dr. Filippo Giancotti  kindly provided expression constructs encoding wild-type or mutant, truncated human β4 subunits. These were engineered in the eukaryotic expression vector pRC-CMV  as described ; pRC-CMV6) were grown to sub-confluence in 10 cm tissue culture dishes and serum-starved (18 h) prior to use. Cells were washed with PBS (3×), treated with varying BMD122 concentrations for 24 h, and subsequently stimulated with 3E1 antibody for 5, 15, 30, 60 and 90 min. For DNA isolation, cells from each time point were harvested and lysed with lysis buffer (200 μl) for 5 min, clarified at 500 × g (5 min), and the resulting pellet was re-extracted using 200 μl lysis buffer (2 min) and re-clarified. Supernatants were pooled and treated with SDS (1 %) and DNase-free RNase (5 mg/ml)  for 2 h (56 °C), followed by proteinase K (2.5 mg/ml)  treatment for 2 h (37 °C). Finally, samples were extracted (1x) with alkaline phenol/chloroform/isoamyl alcohol (25:24:1) and DNA was precipitated with 0.3 M sodium acetate (pH 5.2) and ethanol. DNA laddering was assayed from equal numbers of cells, or 20 μg resolved on a 1.2 % agarose gel followed by ethidium bromide staining.Cells (2.5×105/ml) in DMEM-0.1 % BSA. Antibodies to β4 integrin were preincubated with aliquots of cells for 20 min prior to seeding. EGF (1 ng/ml) was added to the lower chamber as a chemoattractant. The final concentration of 12-LOX pharmacological inhibitors added to the lower chambers was: 10 μM CDC or baicalein, or 20 μM BMD122. All conditions were tested in triplicate. After 3 h, inserts were fixed in a Quick-Fix solution, double-stained with hematoxylin and eosin (HE), and mounted for observation and counting. The number of migrated cells (12 fields × 100) was counted in a double-blind manner. Alternately, inserts with 8 μm pores  were coated with 100 ul of phenol red-free, basement membrane and matrix growth factor-reduced Matrigel  . Inserts were seeded from confluent, serum-starved (overnight) A431 cells (5 × 105) in 0.5 ml serum-free media. Where noted, cells were pre-treated with 25 μM BMD122 for 1 h prior to 30 min treatments with the following: 3E1 antibody (3 ug/well) or the natural ligand, laminin (10 μg/ml). The lower chamber contained serum-free medium with EGF (2 ng/ml), and complete media with or without serum served as positive and negative controls, respectively. After 24 h, transmigrant cells on the underside of the insert were stained with Azure A&B/Eosin Y using the Diff Quick Stain Kit  and washed twice with distilled water. After removing residual, non-migrated cells, membranes were cut from the inserts, dissolved in 10 % acetic acid and assayed for dye content at an absorbance of OD570. Results are the mean of three samples.Modified Boyden chambers  were coated with human laminin  on the upper and lower surfaces, and seeded with A431 cells (5x10Isolated RNA (2 μg)  was reverse-transcribed  for real-time PCR  and GAPDH primers; Applied Biosystems, Foster City, CA). All sample reactions were run in triplicate on the AB 7500 Fast Real Time PCR System. Relative expression of 12-LOX was quantified by the Ct value measured against the internal standard GAPDH using the 7500 Fast System SDS Software v1.4.0 (Applied Biosystems).


In the nearly planar 2-pentyloxybenzamide molecule, there is an intramolecular N—H⋯O hydrogen bond involving one amide proton and the ether oxygen. In the crystal, pairs of N—H⋯O hydrogen bonds organize molecules into inversion dimers lying in two planes, (121) and  and 5.60 (2)° with the amide group and the pentyloxy group mean planes, respectively. In the crystal, molecules are linked by pairs of N—H⋯O hydrogen bonds, forming inversion dimers with their molecular planes parallel, but at an offset of 0.45 (1) Å to each other. These dimers are ordered into two types of symmetry-related columns extended along the a axis, with the mean plane of one set of dimers in a column approximately parallel to (121) and the other in a column approximately parallel to (1-21). The two planes form a dihedral angle of 85.31 (2)°, and are linked via C—H⋯O hydrogen bonds and C—H⋯π interactions, forming a three-dimensional framework structure.In the title molecule, C Other substituted 2-alkoxybenzamides have been found to be antagonists of chemotherapy-induced nausea  receptor antagonists  hydrogen bonds forming inversion dimers  of the 2-pentyloxybenzamide forms an angle of 25.29 (2)° with the column axis.In the crystal, molecules are linked by pairs of N-H⋯O  and In contrast, in 2-methoxybenzamide and in 2-(prop-2-enyloxy)benzamide the intermolecular N—H⋯O hydrogen bonds involving the amide groups do not lead to pair formation but generate n-amyl iodide  was added dropwise. The solution was stirred for 12 h at rt. It was then poured into water (200 ml) and extracted with chloroform (3 × 75 ml). The organic phase was dried over anhydrous MgSO4, concentrated in vacuo, and the residue was subjected to column chromatography on silica gel (CHCl3/MtBE/hexane v/v/v 1:1:1) to give the title compound  as colourless crystals (m.p. 362 K). IR  νmax 3434, 3168, 2948, 2868, 1664, 1593, 1387, 1232, 1164, 1018, 832, 788, 765, 575; 1H NMR  0.93 , 1.38–1.48 , 1.84–1.89 , 4.11 ; 6.03 , 6.96 , 7.03–7.07 , 7.42–7.46 , 7.85 , 8.20 , 13C NMR  14.0 (CH3), 22.4 (CH2), 28.2 (CH2), 28.9 (CH2), 69.1 (OCH2), 112.2 (CH), 120.7 (Cquat), 121.0 (CH), 132.5 (CH), 133.3 (Cquat), 157.4 (Cquat), 167.2 .The preparation of the title compound follows a Williamson ether synthesis using DMSO as solvent, analogous to a general procedure  = xUeq(C), where x = 1.5 for methyl and = 1.2 for other H-atoms. The N-bound H atoms were located in a difference electron-density map and freely refined.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S1600536814020571/su2779sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S1600536814020571/su2779Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814020571/su2779Isup3.cmlSupporting information file. DOI: 1024316CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Lewy body and Alzheimer-type pathologies often co-exist.Several studies suggest a synergistic relationship between amyloid-β (Aβ)and α-synuclein (α-syn) accumulation. We have explored the relationshipbetween Aβ accumulation and the phosphorylation of α-syn at serine-129(pSer129 α-syn), in post-mortem human brain tissue and in SH-SY5Yneuroblastoma cells transfected to overexpress human α-syn.We measured levels of Aβ40, Aβ42, α-syn and pSer129 α-syn bysandwich enzyme-linked immunosorbent assay, in soluble and insolublefractions of midfrontal, cingulate and parahippocampal cortex andthalamus, from cases of Parkinson’s disease (PD) with  andwithout dementia , dementia with Lewy bodies and age-matched controls (n = 17). We also examined the relationship ofthese measurements to cognitive decline, as measured by time-to-dementiaand the mini-mental state examination (MMSE) score in the PD patients,and to Braak tangle stage.In most brain regions, the concentration of insolublepSer129 α-syn correlated positively, and soluble pSer129 α-synnegatively, with the levels of soluble and insoluble Aβ. InsolublepSer129 α-syn also correlated positively with Braak stage. In mostregions, the levels of insoluble and soluble Aβ and the proportion ofinsoluble α-syn that was phosphorylated at Ser129 were significantlyhigher in the PD and DLB groups than the controls, and higher in the PDDand DLB groups than the PDND brains. In PD, the MMSE score correlatednegatively with the level of insoluble pSer129 α-syn. Exposure of SH-SY5Ycells to aggregated Aβ42 significantly increased the proportion of α-synthat was phosphorylated at Ser129 .Together, these data show that the concentration of pSer129α-syn in brain tissue homogenates is directly related to the level of Aβand Braak tangle stage, and predicts cognitive status in Lewy bodydiseases.The online version of this article (doi:10.1186/s13195-014-0077-y) contains supplementary material, which is available toauthorized users. Alzheimer’s disease (AD), Parkinson’s disease (PD) anddementia with Lewy bodies (DLB) are the most common age-relatedneurodegenerative diseases and together account for 80% to 90% of patientswith dementia ,2. The pIn PD and DLB, the number of cortical α-syn aggregates issignificantly higher in patients who have Aβ plaques in the cortex,26 and αet al. [in vitro.The predominant modification of α-syn in Lewy body diseases isphosphorylation at Ser129 ,35.Appret al.  found, tet al. -49, andWe studied 35 cases of PD (23 PDND and 12 PDD) from theQueen Square Brain Bank (QSBB) for Neurological Disorders, UCL Instituteof Neurology, London, and 10 cases of DLB and 17 age-matched controlsfrom the South West Dementia Brain Bank (SWDBB), University of Bristol samples of midfrontal, cingulate andparahippocampal cortex and thalamus were sequentially extracted in 1%NP-40 buffer , 2 mM 1,10 phenanthroline) aspreviously described for Aβ measurements in human post-mortem tissue,48,49,52Total α-syn level was determined by sandwich ELISA. Mousemonoclonal anti-α-syn antibody was coated onto a NUNC Maxisorp 96-well plate overnight at RT. The platewas washed in PBS/0.01% tween-20 and blocked for 1.5 hours in 1% BSA/PBS.Tissue samples (insoluble and soluble extracts diluted 1:200 in PBS) wereadded for two hours at RT with constant shaking. The plate was rinsed,tapped dry and biotinylated polyclonal anti-α-synuclein  diluted in PBS was added for two hours at RT. Theplate was rinsed and tapped dry, streptavidin-horseradish peroxidase(HRP)  was added for 20 minutes and, afterfurther washing, chromogenic substrate  was addedfor 20 minutes in the dark. The reaction was stopped with 2 N sulfuricacid and absorbance at 450 nM read in a FLUOstar Optima plate reader . Total α-syn levels were interpolated frommeasurements made on serial dilutions of recombinant human α-syn rangingfrom 62.5 to 0.98 ng/ml . Measurementsfor each sample were repeated in duplicate.Mouse monoclonal anti-α-syn antibody  was coated onto a NUNC Maxisorp 96-well plate overnight atRT. The plate was washed in PBS/0.01% tween-20 and blocked for two hoursin 1% BSA/PBS. Tissue samples  were added for five hours at RT withconstant shaking. The plate was rinsed and tapped dry and anti-pSer129α-syn  diluted in PBS was added and leftto incubate at 4°C overnight. Following washing of the plate,biotinylated horse anti-rabbit antibody  diluted in PBS with 0.01% tween-20 was added for onehour at RT. The plate was rinsed and tapped dry, streptavidin-HRP wasadded for one hour followed by chromogenic substrate for 20 minutes inthe dark. The reaction was stopped with 2 N sulfuric acid and absorbanceat 450 nM read in a FLUOstar Optima plate reader (BMG Labtech). Theconcentration of pSer129 α-syn was determined as described previously, by inteet al.[et al. [We conducted a preliminary study to confirm the specificityof the pSer129 α-syn antibody. Full-length recombinant human α-syn was incubated with casein kinase I (CKI) or casein kinase II(CKII)  forone hour at 30°C in the presence of 200 μM ATP (New England Biolabs). As a cet al.[et al. [The pSer129 α-syn antibody labelled α-syn followingincubation with CKII, and to a lesser extent CKI, but did not labelrecombinant α-syn that had not been phosphorylated with CKI or CKII. Incontrast, a non-phosphorylation-specific α-syn antibody (BD Biosciences)detected all forms of α-syn, and a pSer87-specific α-syn antibodydetected a signal only after incubation of α-syn with CKI . These The level of Aβ40 was measured in post-mortem brain tissuesamples by sandwich ELISA as described ,56.HighThe level of Aβ42 was measured in post-mortem brain tissuesamples by sandwich ELISA as outlined above with a few modifications.Anti-human Aβ1-42  was used as the captureantibody. Tissue samples  were incubated at RT for four hours. Biotinylatedanti-human Aβ  diluted in PBS wasused for detection and incubated overnight at 4°C. Following washing,rinsing and drying, streptavidin-HRP was added to the plate for one hourand chromogenic substrate for 20 minutes in the dark. Aβ1-42concentration in brain tissue was interpolated from a standard curvegenerated by serial dilution  of recombinant humanAβ1–42 (Sigma Aldrich). Each sample was assayed in duplicate. The Aβ1-42ELISA did not detect Aβ1-40, and the Aβ1-40 ELISA did not detectAβ1-42.P values), enabling validcomparisons to be made between brains and also between cohorts.Intra-assay and inter-assay coefficients of variation werecalculated for the ELISAs as well as spike and recovery tests , Aβ1-40 , pSer129 α-syn  and α-syn  by use of a standard streptavidin-biotin-HRPimmunohistochemistry protocol . The extSNCA cDNA under the control of acytomegalovirus (CMV) promoter. Transfection was carried out withTransFast , followed by selection of clones(and their subsequent maintenance) in culture medium containing0.3 mg/ml G418 . The culturemedium for SH-SY5Y cells, either untransfected or stably expressing humanwild-type α-syn, consisted of 42% vol/vol Ham’s F12 nutrient mixture(F12) (Sigma) and 42% vol/vol Eagle’s minimum essential medium (Sigma),supplemented with 15% vol/vol fetal calf serum (Sigma), 2 mM L-glutamine(Sigma), 1% vol/vol non-essential amino acids solution (Sigma), 20units/mL penicillin, 20 mg/mL streptomycin (Sigma) and 250 ng/mLamphotericin B (Life Technologies) at 37°C in 5%CO2 (21% O2).SH-SY5Y neuroblastoma cells were transfected with apCDNA3.1 vector  containing wild-type humanBefore treatment with Aβ, the culture medium was replacedwith serum-free medium  for 24 hous.Aβ solutions were also prepared 24 hours in advance. Stock solutions of1 mM Aβ42 and Aβ40  in 35%acetonitrile were diluted in serum-free medium at 1 μM and 10 μM. The Aβwas either left overnight to aggregate at 26°C for 24 hours (aspreviously described )or immeCells were incubated with Dulbecco’s PBS without calciumchloride and magnesium chloride (Sigma-Aldrich) at 37°C for five minutesand then removed from the flask, transferred into a Falcon tube, and spunfor three minutes at 13,000 rpm. The cells were washed in PBS and lysedin 100 μl non-denaturing proprietary cell lysis buffer  according to the manufacturer’s guidelines, and spun at13,000 rpm for 15 minutes at 4°C. Cell supernatants (soluble fraction)were removed and stored at −80°C until used. A total of 6 M GuHCl(100 μl) was added to the remaining insoluble pellet and left at RT for1.5 hours (insoluble fraction) before the tube was stored at−80°C.invitro measurements on cells exposed to differentconcentrations of Aβ during the same experiment. For variables that werenot normally distributed even after transformation, the Kruskall-Wallistest was used, with Dunn’s test for pairwise intergroup comparisons.Pearson or Spearman analysis was used as appropriate to assess thecorrelation between pairs of variables. Statistical tests were performedusing GraphPad Prism v5. P-values<0.05 were considered statistically significant.Whenever possible, parametric statistical tests were usedfor comparisons between groups : analysis ofvariance (ANOVA) with Dunnett’s test for pairwise intergroup comparisons,or repeated measures ANOVA for the analysis of P = 0.003; soluble Aβ42: r = 0.287, P = 0.024) and thalamus . Significantpositive correlations were also found between insoluble pSer129 α-syn andinsoluble Aβ42 in the cingulate  and parahippocampal cortex  as well as betweeninsoluble pSer129 α-syn and insoluble Aβ40 in the midfrontal  and cingulate cortex. Significantnegative correlations between soluble pSer129 α-syn and soluble Aβ42 werefound in the cingulate cortex and thalamus . In contrast,a significant positive correlation was observed between soluble Aβ42 andsoluble pSer129 α-syn in the midfrontal cortex . Significant negativecorrelations between soluble Aβ40 and soluble pSer129 α-syn were found inthree of four regions . Significant negative correlations were alsofound between soluble pSer129 α-syn and insoluble Aβ42 in the midfrontalcortex and thalamus . A significant positivecorrelation between soluble pSer129 α-syn and insoluble Aβ40 was alsofound in the parahippocampus .In most regions the level of insoluble pSer129 α-synincreased as the levels of Aβ40 and Aβ42 increased  . Soluble pSer129 α-synlevels correlated negatively with the Braak stage in the cingulate regiononly .The insoluble pSer129 α-syn level correlated positivelywith the Braak tangle stage only in the mid-frontal cortexFigure  . Absolute proteinlevels of pSer129 α-syn are displayed in Additional file In most regions, the proportion of insoluble α-syn that wasphosphorylated at Ser129 was significantly higher in the PDD and DLBgroups than the controls Figure  and in sP = 0.017; cingulate:r = −0.816, P <0.0001;parahippocampal cortex: r = −0.752, P= 0.0003). Furthermore, in the midfrontal region there was a significantnegative correlation between the MMSE score and both insoluble Aβ42 and total insoluble α-syn . No significant correlations wereobserved between MMSE score and soluble Aβ42, Aβ40, total α-syn orpSer129 α-syn. Time to dementia correlated negatively with soluble Aβ40in this region but did not show any other significant correlations (data notshown).In all regions apart from the thalamus, the MMSE scorecorrelated negatively with the level of insoluble pSer129 α-synFigure  (midfronP = 0.06) between the concentration ofaggregated Aβ and the percentage of α-syn phosphorylated at Ser129. Inthe soluble fraction, the percentage of α-syn that was phosphorylated atSer129 was much lower and tended to decline after exposure to aggregatedAβ1-42 and Aβ1-40 but not after exposure to fresh Aβ significantly increased the percentage of α-syn in the insoluble fractionthat was phosphorylated at Ser129. Ser129 phosphorylation was also higherafter exposure to soluble 10 μM Aβ1-42 and aggregated (but not fresh)10 μM Aβ1-40 but these increases did not reach statistical significance significant correlationsbetween phosphorylation of α-syn at Ser129 and the amount of soluble andinsoluble Aβ; (2) significant correlations between phosphorylation of α-synat Ser129 and Braak stage; (3) higher levels of soluble and insoluble Aβ inPD and DLB than controls, and PDD and DLB than PDND; and (4) a higherproportion of α-syn phosphorylated at Ser129 in PD and DLB than controls,and PDD and DLB than PDND. Our study also showed that the proportion ofα-syn phosphorylated at Ser129 correlated with ante-mortem MMSE. Lastly, ouret al. [et al. [Our biochemical studies support previous immunohistochemicalfindings of a positive correlation between insoluble α-syn and Aβ in Lewybody disease ,26,62-64et al.  reportedet al. ,66. Furtet al. ,21-23.Tet al. . Other e [et al. , of enhaet al. [Obi et al.  demonstret al. -70. Somein vitro, bymultidimensional nuclear magnetic resonance (NMR) spectroscopy [etal. [in vitro [Direct molecular interaction between α-syn and Aβ wasdemonstrated troscopy . Aβ42 in [etal.  whoobsein vitro . In keepIn vitro studies have shown that α-syncan be phosphorylated at Ser129 by CKI, CKII [KI, CKII , severalKI, CKII , leucineKI, CKII and PoloKI, CKII ,76. TheKI, CKII , raisingKI, CKII -80.etal. [More than 90% of α-syn in Lewy bodies and neurites isphosphorylated at Ser129 ,35. Theetal. , basedoetal. ,44 and setal. . The accetal. ,81. Theetal. -84. Our et al. [We have found a significant negative correlation between thelevel of insoluble pSer129 α-syn and the MMSE score. This supports previouswork suggesting that Ser129 phosphorylation increases the neurotoxicity ofα-syn and is detrimental to cognitive function. Sato et al. showed tet al. . Thesemet al. . AlthougOur findings in this study, the first to examine therelationship between α-syn, pSer129, Aβ1-40 and Aβ1-42 levels in humanpost-mortem brain tissue by sandwich ELISA, support the existence of apathogenic relationship between the accumulation of Aβ, particularly Aβ42,and the phosphorylation of α-syn at Ser129, increasing the severity of Lewybody disease and the likelihood of dementia. Further investigations arerequired to determine the precise biochemical pathways responsible for thisinteraction, the relative contributions of different processes (includingAβ-associated Ser129 phosphorylation of α-syn and α-syn-associatedphosphorylation of tau) on the development of combined AD and Lewy bodypathology and the progression of neurodegeneration, and also the possibleinfluence of Aβ on other potential sites of α-syn phosphorylation.


The polymeric sheets alternate with layers of counter ions, free ligands and solvent molecules.[Cd(C 2N:N′]cadmium] bis(perchlorate) 1,5-bis(pyridin-4-yl)pentane ethanol monosolvate], [Cd(C15H18N2)2(H2O)2](ClO4)2·C15H18N2·C2H6O, is a layered coordination polymer built up from highly squashed 44 nets in which the octahedral trans-CdO2N4 nodes (Cd site symmetry -1) are linked by the bifunctional ligands, forming infinite (110) sheets. The cationic sheets are charge-balanced by interlayer perchlorate ions. A free 1,5-bis(pyridin-4-yl)pentane molecule and an ethanol molecule of crystallization are also found in the intersheet region. A number of O—H⋯O, O—H⋯N and C—H⋯O hydrogen bonds help to consolidate the layered structure.The title compound, poly[[diaquabis[1,5-bis(pyridin-4-yl)pentane-κ The neutral bridging ligand necessitates the presence of perchlorate counter-ions , which exert an important influence on the structure.The most popular linking ligands in metal-organic frameworks (MOFs) are probably multi-functional carboxylates , three 1,5-bis(pyridin-4-yl)pentane  molecules, two perchlorate ions, two water molecules and one ethanol molecule 2L2]2+n.The asymmetric unit of (I)le Fig. 1. The cadtrans-CdO2N4 octahedral coordination geometries  are 14.4350 (6) and 14.7807 (6) Å. The shortest non-bonded Cd1⋯Cd1 and Cd2⋯Cd2 separations across a squashed 44 square are both 11.0921 (5) Å. It is interesting that the shortest metal–metal distances in (I)Both cadmium ions adopt almost regular s Table 1 arising s Table 1 in valengaaa  conformation (reading from the N11 ring to the N12 ring). Cd1 is displaced by 0.69 (1) Å from the N11 ring plane and Cd2 is displaced by −0.26 (1) Å from the N12 plane. In the N21 ligand molecule, the dihedral angle between the pyridine rings is 75.2 (4)° and the alkyl-chain conformation is aaag (in the sense of the N21 ring to the N22 ring). The displacement of Cd1 from the N21 ring is 0.42 (1) Å and the displacement of Cd2 from the N22 ring is −0.58 (1) Å. The shortest out-and-back pathway from any metal atom to itself encompasses no fewer than 56 atoms .For the N11 ligand molecule, the dihedral angle between the N11 and N12 rings is 77.8 (4)° and the alkyl chain adopts a ggaa 2O)2L2]n sheets propagate in the (110) plane  hydrogen bond also occurs. A number of C—H⋯O interactions are also observed (mean H⋯O = 2.54 Å): see Table 2In the crystal, the infinite [Cd2(NO3)2]n,  8(NO3)8]n·2nH2O, (II), Only four ‘hits’ for crystal structures containing 1,5-bis(pyridin-4-yl)pentane were obtained from a search of Version 5.31 (last update February 2014) of the Cambridge Structural Database 2·xH2O  in water (5 ml). The solution was left to stand for two weeks during which time colourless blocks of (I)−1 ν = 3469 s, 3422 s, 2932 s, 2858 s, 1513 s, 1427 s, 1226 s, 1094 s, 1012 w, 842 w, 800 w, 624 s and 512 w.1,5-Bis(pyridin-4-yl)pentane  = 1.2Ueq or 1.5Ueq(methyl C) was applied in all cases. Crystal data, data collection and structure refinement details are summarized in Table 3The O-bound H atoms were located in difference maps and refined as riding atoms in their as-found relative positions. The C-bound H atoms were placed geometrically and refined as riding atoms. The H atoms of the methyl group were allowed to rotate, but not to tip, to best fit the electron density. The constraint 10.1107/S1600536814014779/wm0007sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S1600536814014779/wm0007Isup2.hklStructure factors: contains datablock(s) I. DOI: 1008978CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


This unit comprises two monodentate water O-atom donors and six carboxylate O-atom donors, two of which are also bridging, from the two bidentate chelate levulinate ligands. The complex chains are stabilized by intra- and intermolecular water O—H⋯O hydrogen bonds, forming an overall three-dimensional structure.The crystal structure of calcium levulinate dihydrate forms a one-dimensional coordination polymer based on a CaO 5H7O3)2(H2O)2]n, the Ca2+ ion lies on a twofold rotation axis and is octacoordinated by two aqua ligands and six O atoms from four symmetry-related carboxylate ligands, giving a distorted square-antiprismatic coordination stereochemistry [Ca—O bond-length range = 2.355 (1)–2.599 (1) Å]. The levulinate ligands act both in a bidentate carboxyl O,O′-chelate mode and in a bridging mode through one carboxyl O atom with an inversion-related Ca2+ atom, giving a Ca⋯Ca separation of 4.0326 (7) Å. A coordination polymeric chain structure is generated, extending along the c-axial direction. The coordinating water molecules act as double donors and participate in intra-chain O—H⋯O hydrogen bonds with carboxyl O atoms, and in inter-chain O—H⋯O hydrogen bonds with carbonyl O atoms, thus forming an overall three-dimensional structure.In the title calcium levulinate complex, [Ca(C Furthermore, due to this type of coordination environment, the two levulinate anions are almost perpendicular to each other, with an O2—Ca1—O2iii angle = 75.78 (5)° : C, 39.21; H, 5.92%. 1H NMR (DMSO-d6) δ 2.05 , 2.19 , 2.54 . 13C NMR (DMSO-d6) δ 30.2, 31.5, 37.9, 179.6, 208.9. The single crystals for X-ray crystallographic analysis were grown by allowing a saturated solution of calcium levulinate dihydrate in 20% methanol in water to stand at room temperature for five days.Levulinic acid  was added to a suspension of calcium hydroxide  in 200 mL of deionized water in a beaker. The mixture was boiled with magnetic stirring on a hot plate to form a clear solution, then transferred to an evaporating dish and allowed to crystallize at room temperature. The product was collected under suction filtration, dried at 363 K for 24 h to give 1.455 g of calcium levulinate dihydrate as white needle-shaped crystals in 95% yield. Found: C, 39.02; H, 6.23; calculated for [Ca(CUiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) for other H atoms. The water H atoms were found using a Fourier map and were also allowed to ride in the refinement, O—H = 0.90 Å and with Uiso(H) = 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015006696/zs2328sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989015006696/zs2328Isup2.hklStructure factors: contains datablock(s) I. DOI: 1057749CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Space filling more symmetric than atom positions leads to pronounced orientational disorder for the DABCO ligand.The one-dimensional coordination polymer, self-assembled from bis(acetylacetonato)cobalt(II) units as metal–complex  5H7O2)2(C6H12N2)]n, was obtained as a one-dimensional coordination polymer from bis(acetylacetonato)diaquacobalt(II), [Co(acac)2(OH2)2], and 1,4-diazabicyclo[2.2.2]octane (DABCO), a diamine with good bridging ability and rod-like spacer function. In the chain complex that extends along the c axis, the CoII atom is six-coordinated, the O-donor atoms of the chelating acac ligands occupying the equatorial positions and the bridging DABCO ligands being in trans-axial positions. In the crystal structure, the DABCO ligand is conformationally disordered in a 50:50 manner as a result of its location across a crystallographic mirror plane. The metal–metal distance is very close to that in a related compound exhibiting weak antiferromagnetic exchange between the CoII ions, and the title compound can thus be useful for obtaining more information about the contribution of different bridges to the magnetic coupling between paramagnetic ions.The title compound, [Co(C Self-assembly of coordination polymers from simple building blocks is of considerable interest due to their diverse architectures and potential applications in catalysis and advanced materials, such as magnetic, optic and electronic materials.2O,O′)cobalt(II), [Co(acac)2], have been chosen to design a one-dimensional coordination polymer in which the paramagnetic CoII ions are separated by a distance of 7.2328 (7) Å. This metal–metal distance is very close to the distance of 7.267 (3) Å reported by Ma et al. (20012(pyrazine)]n compound which exhibits weak antiferromagnetic exchange between the CoII ions.In this paper, two simple building blocks, namely 1,4-diazabicyclo[2.2.2]octane (DABCO), a diamine with good bridging ability and rod-like spacer function, and the unsaturated square-planar metal complex biscobalt(II)]-μ-1,4-diazabicyclo[2.2.2]octane-κ2N1:N4], [Co(acac)2(DABCO)]n, (I)Within this context, the title compound connectors and 1,4-diazabicyclo[2.2.2]octane (DABCO) as linkers.In the crystalline state, the title compound, (I)O,O′-chelating donor agent and its metal coordination chemistry is well documented . For DA al. 2009; Uemura  al. 2007].Pnnm space group with the metal atom on a special position with site symmetry ..2/m. The CoII atom shows an octahedral environment defined by four equatorial acac O atoms on a mirror plane, with bond lengths ranging from 2.0299 (10) to 2.0411 (10) Å, and with two N atoms of bridging DABCO groups on a twofold rotation axis in the axial positions at distances of 2.3071 (12) Å is Fig. 2. The ind2(μ-diamine)]n n complexes similar in structure to the title compound, with square-planar [Co(acac)2] units connected by bridging diamine ligands into infinite linear chains, were retrieved from the Cambridge Structural Database 2{μ-1,3-bis(pyridin-4-yl)propane}]n ]n and [Co(acac)2]n 2(H2O)2] was prepared by precipitation of CoCl2·6H2O with aqueous ammonia, followed by solubilization and complexation with acetylacetone. Elemental analysis calculated for [Co(C5H7O2)2(H2O)2] (%): C 40.96, H 6.14; found: C 40.94, H 6.19.[Co(acac)2(H2O)2]  and 1,4-diazabicyclo[2.2.2]octane (DABCO)  were stirred in CH3OH (15 ml) at 333 K for 1 h. The pink precipitate which formed was collected by filtration and redissolved in dimethyl sulfoxide . Elemental analysis calculated for [Co(acac)2(DABCO)] (%): C 52.04, H 7.05, N 7.59; found: C 51.63, H 7.39, N 7.41. Layering the solution of the complex in DMSO with CH3OH at 293 K gave pale-pink crystals suitable for X-ray single-crystal analysis.[Co(acac)Elemental analyses were carried out on a Heraeus CHNO Rapid apparatus .et al., 1993Crystal data, data collection and structure refinement details are summarized in Table 1Uiso(H) = 1.5Ueq(C) for methyl H atoms and Uiso(H) = 1.2Ueq(C) otherwise. For consistency, we opted to calculate the positions of the DABCO H atoms and fix them in their idealized positions. Due to the fact that the acac ligand lies on a mirror plane, the acac methyl groups are therefore equally disordered over two orientations.H atoms attached to C atoms were calculated, introduced in their idealized positions and treated as riding, with C—H = 0.95 Å and 10.1107/S205698901600428X/wm5278sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S205698901600428X/wm5278Isup2.hklStructure factors: contains datablock(s) I. DOI: 1454848CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In both compounds, the bond lengths in the H 8H8N3S2+·Cl−, (I), and salt hydrate, C8H7ClN3S2+·Cl−·H2O, (II), are described. The heterocyclic ring in (I) is statistically planar and forms a dihedral angle of 9.05 (12)° with the pendant phenyl ring. The comparable angle in (II) is 15.60 (12)°, indicating a greater twist in this cation. An evaluation of the bond lengths in the H2N—C—N—C—N sequence of each cation indicates significant delocalization of π-electron density over these atoms. The common feature of the crystal packing in (I) and (II) is the formation of charge-assisted amino-N—H⋯Cl− hydrogen bonds, leading to helical chains in (I) and zigzag chains in (II). In (I), these are linked by chains mediated by charge-assisted iminium-N+—H⋯Cl− hydrogen bonds into a three-dimensional architecture. In (II), the chains are linked into a layer by charge-assisted water-O—H⋯Cl− and water-O—H⋯O(water) hydrogen bonds with charge-assisted iminium-N+—H⋯O(water) hydrogen bonds providing the connections between the layers to generate the three-dimensional packing. In (II), the chloride anion and water molecules are resolved into two proximate sites with the major component being present with a site occupancy factor of 0.9327 (18).The crystal and molecular structures of the title salt, C While earlier work focussed on supramolecular aggregation patterns =N(p-tolyl)] compounds prove to be very potent against Gram-positive bacteria =N(aryl)] compounds exhibit significant cytotoxicity and kill cancer cells by initiating a variety of apoptotic pathways  complexes 8H7ClN3S2]Cl·H2O (II)The title salts were isolated as a part of a research programme into the crystal engineering aspects and biological potential of phosphanegold(I) carbonimidothioates, The asymmetric unit of (I)The asymmetric unit of (II)Fig. 3Geometric parameters characterizing the intermolecular interactions operating in the crystal structures of (I)− and N+—H⋯Cl− hydrogen bonds are crucial in establishing the three-dimensional architecture in the crystal structure of (I)a axis connected through hydrogen bonds to rows of chloride ions, also aligned along the a axis. As illustrated in Fig. 4− hydrogen bonds lead to helical chains along [100], being generated by 21 screw symmetry. The chains are linked to neighbouring chains by charge-assisted iminium-N+—H⋯Cl− hydrogen bonds, that in themselves lead to chains aligned along [011]. In this way, a three-dimensional architecture is constructed as shown in projection in Fig. 5The presence of charge-assisted N—H⋯Cl− hydrogen bonds while the iminium-H atom forms a charge-assisted N+—H⋯O hydrogen bond to the water molecule of crystallization. The water molecule also forms two donor interactions, one to another water molecule and the second, charge-assisted, to the chloride anion. Hence, all donor atoms participate in the hydrogen-bonding scheme and each of the chloride and water species forms three hydrogen bonds. A diagram showing the detail of the hydrogen bonding is shown in Fig. 6− bridges clearly persist, as for (I)c axis. As pairs of water molecules are linked via water-O—H⋯O(water) hydrogen bonds across a centre of inversion and each forms a charge-assisted water-O—H⋯Cl− hydrogen bond, the water molecules form links between the zigzag chains resulting in supramolecular layers. Finally, the water molecules accept charge-assisted imino-N+—H⋯O(water) hydrogen bonds, providing links between the layers so that a three-dimensional architecture ensues. As seen from Fig. 7i contacts of 3.3510 (10) Å which occur within layers rather than between layers; symmetry operation (ii): 1 − x, y, −z.A more complicated pattern of hydrogen bonding occurs in the crystal structure of (II)i.e. 5-(dimethylamino)-3-(phenylimino)-1,2,4-dithiazole, characterized in its 1:1 co-crystal with 2-(dimethylcarboxamido-imino)benzothiazole, (III) in the scheme below, has been reported   To thiourea  in acetonitrile (20 ml) was added 50% w/v NaOH  and phenyl isothiocyanate . The resulting mixture was stirred for 4 h at 323 K. 5 M HCl  was added and the mixture was stirred for another 1 h. The final product was extracted using chloroform (200 ml). The powder that formed after 2 weeks was re-dissolved in dichoromethane/acetonitrile , yielding yellow prisms after 3 weeks. Yield: 0.627 g (51%). M.p. 492–493 K. 1H NMR : 13.37 , 10.73 , 10.66 , 7.74 , 7.45 , 7.27 . 13C NMR : 182.9 [SC(=N)N], 176.1 [C(NH2)], 138.5 (Cipso), 129.7 (Cmeta), 126.5 (Cpara) 121.4 (Cortho). IR (cm−1): 3414 (m) (N—H), ν 3007 (m) (C—H), ν 1248 (s) (C—N).Synthesis of (II) The p-chloro derivative (II)1H NMR : 13.51 , 10.72 , 10.41 , 7.77 , 7.52 , 3.48 . 13C NMR : 183.0 [SC(=N)N], 176.1 [CNH2], 137.4 (Cipso), 130.3 (Cpara), 129.6 (Cmeta), 122.9 (Cortho). IR (cm−1): ν 2965 (br) (O—H), ν 1250 (s) (C—N).Uiso(H) set to 1.2Ueq(C). The N-bound H-atoms were located in a difference Fourier map but were refined with a distance restraint of N—H = 0.88±0.01 Å, and with Uiso(H) set to 1.2Ueq(N). For (I)i.e. (020), was omitted from the final cycles of refinement. For (II)Uiso(H) = 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989015016655/hb7500sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989015016655/hb7500Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015016655/hb7500IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989015016655/hb7500Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015016655/hb7500IIsup5.cmlSupporting information file. DOI: 1422604, 1422603CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal structure of salt (I), one of the –NH H atoms of the imidazole ring shows half-occupancy, hence only every second molecule is in its cationic form. The nitrate anion in this structure lies on a twofold rotation axis. The neutral 2-methylimidazole molecule and the 2-methyl-1H-imidazol-3-ium cation interact through N—H⋯N hydrogen bonds to form [(C4H6N2)⋯(C4H7N2)+] pairs. These pairs are linked with two nitrate anions on both sides through bifurcated N—H⋯ hydrogen bonds into chains running parallel to [001]. In the crystal structure of salt (II), the C4H7N2+ cation and the NO3− anion are both located on a mirror plane, leading to a statistical disorder of the methyl H atoms. The cations and anions again interact through bifurcated N—H⋯ hydrogen bonds, giving rise to the formation of chains consisting of alternating anions and cations parallel to [100].The title salts, C IV complexes, crystals of the salt C4H7N2+·NO3−, (II)4H6N2·C4H7N2+·NO3−, and report the two structures in this communication. While targeting the synthesis of new SnH-imidazol-3-ium cation and a neutral 2-methylimidazole molecule in the crystal applying symmetry operation (i) 1 − x, y, z  z Fig. 1. In the s Table 1. The imion Fig. 2, both lyH-imidazol-3-ium cations and for the neutral 2-methylimidazole molecule, the N—C distances involving C2, the C atom that carries the methyl group, are equal within 0.01 Å, and their values are consistent with double-bond character, as previously observed  reflect a perfect trigonal–planar geometry for each of the nitrate anions. For the 2-methyl-1H-imidazol-3-ium cation through N—H⋯N hydrogen bonds, forming a [(C4H6N2)⋯(C4H7N2)+] pair ir Fig. 1. Such pair Fig. 1, leadingir Fig. 1.H-imidazol-3-ium cations and the nitrate anions are alternately linked by bifurcated N—H⋯ hydrogen bonds s Table 2, leadings Table 2.In the two structures, the stability between the chains is dominated by electrostatic interactions.H-imidazol-3-ium nitrate -1H-imidazol-3-ium nitrate  and were used as received. Single crystals suitable for X-ray studies of (II)vs 0.47 e Å−3 for an occupancy factor of 1 and 0.5, respectively). For (II)Uiso(H) = 1.2 and 1.5Ueq(C), respectively. As a result of the mirror symmetry of the 2-methyl-1H-imidazol-3-ium cation, the methyl H atoms are statistically disordered over two positions. H atoms bound to N atoms were located from a difference Fourier map and were freely refined.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989016003789/wm5276sup1.cifCrystal structure: contains datablock(s) global, II, I. DOI: 10.1107/S2056989016003789/wm5276Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989016003789/wm5276IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989016003789/wm5276Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016003789/wm5276IIsup5.cmlSupporting information file. DOI: 1458009, 1458008CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II atoms in two N-heterocyclic carbene(NHC)/halogenide complexes show distorted square-planar coordination environments. In one complex, two NHC ligands are present, and the second complex contains an auxiliary triphenylphosphane ligand.The Pd 19H28N2)2I2], (I), and [Pd(C19H28N2)Cl2(C18H15P)], (II), show a distorted square-planar coordination around the PdII atoms with two halogenide  ligands each and two N-heterocyclic carbene (NHC) ligands in (I) or one NHC and one triphenylphosphane ligand in (II). The deviations of the PdII atoms from the L2Hal2 best plane (L = NHC or triphenylphosphane ligand) are 0.206 (1) Å for (I) and 0.052 (1) Å for (II). The crystal packings exhibit intermolecular C—H⋯Hal hydrogen bonds.The molecular structures of the chiral title compounds, [Pd(C N-substituents exhibit a larger proximity to the metal atom, which can be an advantage for the transfer of chirality from the ligand to the product during a catalytic reaction or for the reductive elimination during the catalytic cycle  ligands differ from the extensively reported five-membered analogues in several aspects. As a result of the increased N—C—N angle, the  al. 2004 and Poul al. 2014.S,5S)-4-mesityl-1,2,8,8-tetramethyl-2,4-diazabicyclo[3.2.1]octan-3-ylidene]} with two NHC-ligands in Pd(C19H28N2)I2, (I)19H28N2)(C18H15P)Cl2, (II)We report herein on the syntheses and crystal structures of two N-heterocyclic-carbene (NHC)–Pd complexes {the chiral carbene being  ] Table 3.A⋯Cl2 hydrogen bonds link molecules into endless rows running parallel to [010]. Additionally, an intramolecular C42—H42A⋯Cl2 bond is present t Table 4.et al. -4-mesityl-1,2,8,8-tetramethyl-2,4-diazabicyclo-[3.2.1]-oct-2-en-2-ium iodide  and THF (3 ml) for (I)c = 0.5 mol/l) for (I)c = 0.5 mol/l for (II)2(PPh3)2  for (I)2(PPh3)2  for (II)2I2 (I)3)Cl2 (II)The synthesis of the carbene precursor has been described by Koppenwallner  al. 2015. The titUiso(H) = 1.2U(Ceq) or 1.5U(Ceq) (–CH3) and C—H 0.95–1.00 Å. All CH3 hydrogen atoms were allowed to rotate but not to tip.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989015013055/wm5179sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989015013055/wm5179Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015013055/wm5179IIsup3.hklStructure factors: contains datablock(s) II. DOI: 1411191, 1411190CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The dominant interaction in (I) and (II) is an O—H⋯O hydrogen bond across the bicyclic 5,5-ring system between the non-functionalized hydroxy group and the tetrahydrofuran O atom, which appears to influence the envelope conformations of the fused five-membered rings, whereas in (III), the rings have different conformations. A weak intramolecular C—H⋯O interaction appears to influence the degree of tilt of the phenyl ring attached to the 5-position and is different in (I) compared to (II) and (III).The crystal structures of three cyclopenta[ Neosurugatoxin, shown in Scheme 1et al., 1981Neosurugatoxin, Cet al., 1988et al., 1995et al., 1992et al., 1992et al., 1986et al., 1989C in Neosurugatoxin -4-methyl-5-phenylhexahydro-1H-cyclopenta[c]furan-4,5-diol, C14H18O3, (I)R,4S,5S,6aS)-4-benzyloxy-4-methyl-5-phenylhexahydro-1H-cyclopenta[c]furan-5-ol, C21H24O3, (II)R,1bS,4aR,5S,5aR)-5-benzyloxy-5-methyl-5a-phenylhexahydro-2H-oxirenocyclopentafuran, C21H22O3, (III)As part of these studies, we now report the crystal structures of three of these compounds, namely (±)-. The C1/C2/C3/O1/C4 ring has an envelope conformation, with O1 displaced from the mean plane of the carbon atoms (r.m.s. deviation = 0.018 Å) by 0.566 (5) Å. The C1/C2/C5/C6/C7 ring also has an envelope conformation, with C6 displaced from the other atoms (r.m.s. deviation = 0.026 Å) by 0.573 (6) Å. The dihedral angle between the almost planar parts of the rings is 58.3 (2)°: the overall shape could be described as a butterfly, with the flap atoms (O1 and C6) pointing inwards. Atoms O2 and O3 lie to the same face of the ring although there is a significant twist between them [O2—C6—C7—O3 = 46.5 (4)°]. The O2—C6—C7—C8 torsion angle is 164.9 (3)° and the C8—C7—C6—C9 torsion angle is 47.6 (4)°. The dihedral angle between the pendant benzene ring (C9–C14) and C1/C2/C5/C7 is 64.00 (17)°. The molecular structure of (I)o⋯O2 bond closes an S(5) ring. The O2—H2o⋯O1 bond, which bridges across the top of the fused-ring system to generate an S(7) ring, may influence the conformations of the five-membered rings. An intramolecular C10—H10⋯O2 short contact (H⋯O = 2.33 Å) is also present: although the C—H⋯O angle of 100° is extremely small to be regarded as a bond .Compound (I)P21/c, contains one molecule o) grouping forms an intramolecular hydrogen bond (Table 2S(7) ring results. The C10—H10 grouping in (II)The asymmetric unit of (II)le Fig. 2: for easd Table 2 to O1 acP212121. The absolute structure was indeterminate in the present experiment and C1, C2, C5, C6 and C7 in the asymmetric molecule were assigned configurations of S, R, S, S and R, respectively  = 0.307 (10) Å, φ(2) = 232.5 (18)°] such that C2 and C3 are displaced from the O1/C4/C1 plane by −0.22 (2) and 0.29 (2) Å, respectively. The C1/C2/C5/C6/C7 conformation in (III)Compound (III)ly Fig. 3. Based oo⋯O1i  hydrogen bonds (Table 1b-glide symmetry and the chain has a C(6) motif. Long and presumably very weak intermolecular C—H⋯O and C—H⋯π interactions (Tables 2ca 4.97 in (I)In the crystal of (I)s Table 1: the sam Tables 2 and 3 ▸ c]furan skeleton revealed 321 matches; of these, just two had O atoms bonded to the 4- and 5-positions of the fused-ring system, viz.: VALFIX -5-phenyl-1,3,3a,6a-tetrahydro-4H-cyclopenta[c]furan-4-one, Aet al. -4-methyl-5-phenyl-3,3a,4,6a-tetrahydro-1H-cyclopenta[c]furan-4-ol, BBm-CPBA in anhydrous dichloromethane at 273 K yielded (±)--5-methyl-5a-phenylhexahydro-2H-oxirenocyclopentafuran-5-ol, Cet al., 2007C with lithium aluminium hydride in anhydrous tetrahydrofuran -4-methyl-5-phenylhexahydro-1H-cyclopenta[c]furan-4,5-diol, (I)R,4S,5S,6aS)-4-(benzyloxy)-4-methyl-5-phenylhexahydro-1H-cyclopenta[c]furan-5-ol, (II)C using the procedure of Peng & Woerpel -5-(benzyloxy)-5-methyl-5a-phenylhexahydro-2H-oxirenocyclopentafuran, (III)Full synthesis details will be reported in due course, but a summary of the steps followed to prepare (I) al. 2005. Treatme al. 2008 affordedrpel 2003 affordedUiso(H) = 1.2Ueq(carrier) or 1.5Ueq(methyl carrier) was applied in all cases. The methyl H atoms were allowed to rotate, but not to tip, to best fit the electron density. The C16–C21 benzene ring in (III)R-factors obtained, although the structures are clearly resolved with acceptable geometrical precision. The absolute structure of compound (III)Crystal data, data collection and structure refinement details for (I)–(III)10.1107/S2056989015023506/gk2648sup1.cifCrystal structure: contains datablock(s) I, II, III, global. DOI: 10.1107/S2056989015023506/gk2648Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015023506/gk2648IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989015023506/gk2648IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989015023506/gk2648Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015023506/gk2648IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015023506/gk2648IIIsup7.cmlSupporting information file. DOI: 1440873, 1440872, 1440871CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, weak Cu⋯Cl interactions between symmetry-related molecules create a dimerization with a chloride occupying the apical position of the square-pyramidal geometry typical of many copper(II) chloride hetero-scorpionate complexes. 17H19Cl4N3)]ClO4, the CuII ion adopts a distorted square-planar geometry defined by one chloride ligand and the three nitrogen atoms from the bis[(pyridin-2-yl)methyl]amine ligand. The perchlorate counter-ion is disordered over three sets of sites with refined occupancies 0.0634 (17), 0.221 (16) and 0.145 (7). In addition, the hetero-scorpionate arm of the bis[(pyridin-2-yl)methyl]amine ligand is disordered over two sets of sites with refined occupancies 0.839 (2) and 0.161 (2). In the crystal, weak Cu⋯Cl interactions between symmetry-related molecules create a dimerization with a chloride occupying the apical position of the square-pyramidal geometry typical of many copper(II) chloride hetero-scorpionate complexes.In the title compound, [CuCl(C These reactions involve the formation of carbon–carbon bonds through addition of a poly-halogenated saturated hydrocarbon to alkenes  ]/141; Yang et al., 2007II ion sits 0.0922 (4) Å out of the mean basal plane formed by Cl1 and the three coordinating N atoms, giving rise to the distortion from true square-planar geometry. The Cu—Cl1 [2.2519 (8) Å], Cu—N(amine) [2.027 (2) Å], and Cu—N(py) [1.982 (3) and 1.987 (3) Å] bond lengths are in the anticipated range for copper(II) complexes.The title complex, (1) Fig. 1, adopts II atoms  Å] Cu⋯Cl interactions between adjacent molecules creates a dimerization with two Cl atoms bridging the Cums Fig. 2. The intII atom, there are 58 remaining structures. Eighteen of these remaining structures have two bridging Cl ligands with one short axial Cu—Cl bond length (average 2.25 Å) and one long apical Cu—Cl bond length (average 2.72 Å).There are 200 structures with the bis(pyridin-2-ylmethyl)amine ligand coordinating to copper with at least one bound chloride ligand (Groom & Allen 2014Synthesis of 1-butene-bis(pyridin-2-ylmethyl)amine, (B): bis(pyridin-2-ylmethyl) amine (BPMA) precursor (A) was synthesized and purified following literature procedures . The ligand was stored in a septum sealed round-bottom flask under argon gas in a refrigerator. 1H NMR : δ2.31 , δ2.64 , δ3.83 , δ4.97 , δ5.01 , δ5.75 , δ7.13 , δ 7.53 , δ 7.64 , δ8.51 . 13C NMR : δ 159.75, 149.01, 136.38, 135.38, 122.80, 121.88, 117.93, 77.13, 59.90, 57.32. FT–IR (liquid): v (cm−1) = 3066 (w), 2922 (w), 2816 (w), 2158 (s), 1639 (s), 1588 (s), 1361 (s), 994 (w), 756 (s). FT–IR (solid): v (cm−1) = 3394 (w), 3067 (w), 3008 (s), 2923 (w), 2817 (s), 2359 (s), 1619 (s), 1589 (s), 1432 (s).The synthetic procedure is outlined in Fig. 4I(butene-Synthesis of [Cubis4](pyridin-2-ylmethyl)amine)]+, Calculated for C16H19N3Cu 316.0875, found 316.0897 (7 p.p.m.).IIamine)][Cl][ClO4]Synthesis of [ClO4] (C)  was dissolved in 5 mL acetonitrile in a glass vial with a stir bar. Nitrogen gas purged CCl4  was added to the vial producing a bluish-green-colored mixture. The reaction vial was sealed with a plastic cap and allowed to mix for 4 h, then removed from the drybox. Vapour diffusion crystallization at room temperature, incorporating 1 mL of the bluish-green solution with diethyl ether as the external diffusing solvent, produced blue-colored crystals suitable for X-ray analysis.5H7Cl4] arm were restrained to be the same (s.u. 0.01Å). The perchlorate anions were also restrained to have the same geometries (s.u. 0.01 Å). In addition, the sum of the occupancies of the three orientations for the perchlorate anions were restrained to add up to one (s.u. 0.001). All disordered sites were restrained to have similar displacement amplitudes (s.u. 0.01) for atoms overlapping by less than the sum of van der Waals radii. Displacement parameters for the perchlorate anion positions were also restrained to behave relatively isotropic. All non-H atoms were refined with anisotropic displacement parameters. H atoms were included as riding idealized contributors, with C—H = 0.95 (aromatic), 0.99 (sp3 C—R2H2), and 1.00 Å (sp3 C—R3H). The Uiso(H) values were set to 1.2Ueq(C) of the carrier atom. The (002) reflection was omitted from the final refinement because it was partially obscured by the beam stop.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015011792/lh5771sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989015011792/lh5771Isup2.hklStructure factors: contains datablock(s) I. DOI: 1407833CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The metabolic stress-sensing enzyme AMP-activated protein kinase (AMPK) is responsible for regulating metabolism in response to energy supply and demand. Drugs that activate AMPK may be useful in the treatment of metabolic diseases including type 2 diabetes. We have determined the crystal structure of AMPK in complex with its activator 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid (C2), revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results show that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases. AMPK regulates the metabolism and so drugs that activate AMPK might have potential for the treatment of metabolic disease. Here, the authors report the structure of AMPK bound to an activating compound, revealing two binding sites and indicating that dual therapy might be a good drug strategy. Once phosphorylated on α-Thr172 AMPK is further activated by AMP, and AMP sustains AMPK signalling by inhibiting dephosphorylation of α-pThr172 (ref. 167AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase responsible for regulating metabolism in response to energy supply and demand. During metabolic stress the cellular AMP/ATP ratio increases leading to activation of AMPK, which in turn switches off energy-consuming anabolic pathways and switches on catabolic pathways to restore ATP levels. The AMPK αβγ heterotrimer comprises a catalytic α-subunit associated with β and γ regulatory subunits . Adenine2172 ref. . AMP sti172 ref. . The α-r172 ref. 6 is requThere has been keen interest in developing AMPK-activating drugs for potential therapeutic use in treating metabolic diseases including type 2 diabetes, obesity and cardiovascular disease. A number of small-molecule activators have been identified8Another potent small-molecule allosteric activator of AMPK, 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phosphonic acid , was identified by screening an AMP mimetic libraryWe have determined the crystal structure of the C2: AMPK complex, revealing two C2-binding sites in the γ-subunit distinct from nucleotide sites. C2 acts synergistically with the drug A769662 to activate AMPK α1-containing complexes independent of upstream kinases. Our results raise the possibility that dual drug therapies could be effective AMPK-targeting strategies to treat metabolic diseases.We found that C2 activates both AMPK γ1 and γ2 complexes  to a similar degree as AMP, whereas neither C2 nor AMP-activated γ3-containing complexes (α1β1γ3 and α1β2γ3) . Lack of18Since ATP concentration was previously shown to alter AMP dependence−1 mg−1) was co-incubated with combinations of AMP/A769662 (≈671 nmol min−1 mg−1 or≈1100-fold) and C2/A769662 (≈636 nmol min−1 mg−1 or ≈1040-fold), but not with CaMKK2-treated AMPK α1 (−1 mg−1) co-incubated with AMP/A769662 or C2/A769662 was relatively insensitive to dual ligand activation  and solved to an equivalent resolution of 3.02 Å) revealed strong electron density for both AMP-bound heterotrimers in the asymmetric unitOur AMP-bound heterotrimer closely resembles the previously published AMP-bound α2β1γ1 structure (PDB 4CFE)16The two C2 molecules occupy the interface between the CBS-binding sites 1, 3 and 4, with the phosphate group of C2 occupying a similar position to that of AMP-bound phosphate group in sites 1 and 4 . For thiIn both sites the C2 molecule makes six protein-mediated hydrogen bond contacts, while C2 in γ-pSite-4 makes an additional π-stacking interaction with γ-His298 . All of 15−1 mg−1 lysate for γ-T89E and γ-H298E, respectively) in the absence of C2 and AMP compared with γ1 WT (1.4±0.1 nmol min−1 mg−1 lysate), and were insensitive to further activation by either C2 or AMP  in COS7 mammalian cells and measured their sensitivity to C2 or AMP. We initially found that all the γ1 mutants displayed increased but variable Thr172 phosphorylation and basal activities compared with the wild-type (WT) γ1-containing AMPK complexes. We therefore co-expressed the mutants with the phospho-mimetic AMPK α1(T172D), which is more amenable to studying the effect of these γ mutations on allosteric activation 521. C2 a52 or AMP . It is p2 or AMP , while o2 or AMP .et al.67A superposition of the two AMPK heterotrimers reveals two major side-chain conformational changes between AMP and C2 binding. Firstly, the imidazole ring of the aforementioned γ-His298 has flipped 180° and rotated at the Cβ>90° ∼ 6.0 Å) to form a π-stacking interaction with C2 in γ-pSite-4 while simultaneously blocking site 3, mimicking ATP-bound AMPK (PDB 4EAK)1.0 Å to fSecondary structure elements of αRIM2 are crucial for the isoform specificity of C2. Structural comparison of α1 and α2 isoform αRIM2/γ-subunit interactions reveal the α-helical α1RIM2 (PDB 4CFH)In this study we showed that two topographically disparate drug sites on AMPK, one located at the classic α-kinase domain/β-CBM interface (ADaM site) and the other located within the solvent-accessible core of the γ-subunit (γ-pSite-1/γ-pSite-4), can be exploited to achieve synergistic activation of unphosphorylated α1-AMPK independent of AMP and upstream phosphorylation events. This provides a major advance to previous demonstrations of AMPK synergistic activation that have been dependent on AMP. Importantly, identification of C2-binding sites on the γ-subunit represents an entirely unexplored dimension for future rational drug design.Antibodies for pan AMPK α (#2793), FLAG (#2368) and HA (#2367); phosphospecific antibodies for α-pThr172 (#2535) and β-pSer108 (#4181), and λ-phosphatase (#P0753) were from Cell Signaling Technology. β1 antibody (#ab58175) and A769662 (#ab120335) were from Abcam. Anti-rabbit and anti-mouse IgG secondary antibodies fluorescently labelled with IR680 or IR800 dye, respectively, were from LI-COR Biosciences. Glutathione Sepharose 4B was from GE Life Sciences. FLAG peptide (DYKDDDK) was provided by GL Biochem (Shanghai), purified by reversed-phase chromatography and stored as lyophilized powder. All other reagents were purchased from Sigma.Escherichia coli-expressed AMPK preparations were mass verified by time-of-flight (TOF) mass spectrometry. Heterotrimeric human AMPK His6-α2β1γ1 was expressed in E. coli strain Rosetta (DE3) using the pETDuetTM-1 expression system (Novagen) as previously describedTM-1 multiple cloning sites (MCS) 1 (BamH1/Not1) and 2 (MfeI/XhoI), respectively, resulting in incorporation of an N-terminal hexahistidine tag onto α2. cDNA for β1 was inserted into pRSFDuetTM-1 MCS1 (NcoI/ BamHI). Heterotrimeric human AMPK α2/α1 RIM chimaera (α2(1-347)/α1(349-401)/α2(397-end) β1γ1)E. coli strain Rosetta (DE3) using the pETDuetTM-1 and pCOLADuetTM (Novagen). cDNAs for α2/1 chimaera and γ1were sequentially inserted into pETDUETTM-1 multiple cloning sites (MCS) 1 (BamHI/Not1) and 2 (MfeI/XhoI), respectively, resulting in incorporation of an N-terminal hexahistidine tag onto α2/1 chimaera. cDNA for β1 was inserted into pCOLADuetTM-1 MCS1 (NcoI/ BamHI).All mutants were generated using QuikChange Site-Directed Mutagenesis Kits (Stratagene), and constructs were sequence verified. D-1-thiogalactopyranoside, before overnight incubation. Cells were lysed using a precooled EmulsiFlex-C5 homogenizer (Avestin) and AMPK purified using Nickel Sepharose and size exclusion chromatography in 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 2 mM Tris(2-carboxyethyl)phosphine buffer. AMPK was phosphorylated by incubation with CaMKK2 . Protein was re-purified using size exclusion chromatography, concentrated to ∼10 mg ml−1 and flash frozen in liquid nitrogen before storage at −80 °C phosphine).Expression cultures were grown in Luria-Bertani broth and induced at 16 °C with 0.25 mM isopropyl β-−1) was mixed with a twofold molar excess of C2, AMP and S108tide (synthetic peptide: β1(102-117) R115-117, NH2-KLPLTRSHNNFVARRR-COOH) and a 1:1 molar ratio of Staurosporine and A769662, while full-length phosphorylated RIM swap chimaera (α2 1-347/α1 349-401/α2 397-end β1γ1)−1) was mixed with a sixfold molar excess of C2, twofold molar excess of S108tide and a 1:1 molar ratio of Staurosporine and A769662. Crystals from both protein preparations were grown following the previously described method2, 1.0% glucose, 0.001% cocamidopropyl betaine and 0.1 M imidazole (pH 6.2). Protein crystals were incubated with reservoir solution containing an additional 5% glycerol, 5% PEG 400, 5% MPD, 5% sucrose, 5% sorbitol and 5% ethylene glycol for∼2 min before flash-cooling in liquid nitrogen. Data were collected at the Australian Synchrotron MX2 beamline, Melbourne, Australia. Data were processed and integrated using XDShttps://www.globalphasing.com/buster/)https://www.pymol.org/). Data collection and refinement statistics are listed in Full-length myristoylated and phosphorylated α2β1γ1 (4 mg ml2) or CaMKK2   prior to extensive washes with buffer A and elution in buffer A supplemented with 20 mM glutathione. The levels of α-pThr172 and β-pSer108 of treated AMPK complexes were assessed by immunoblot as described previouslyAll mutants were generated using QuikChange Site-Directed Mutagenesis Kits (Stratagene), and constructs were sequence verified. Heterotrimeric AMPK was expressed in COS7 mammalian cells by transient transfection, using FuGENE HD (Promega) transfection reagent according to manufacturer's protocol. Transfected constructs included N-terminal GST-α1 and α2 fusions2-HMRSAMSGLHLVKRR-COOH), 5 mM MgCl2, 200 μM [γ-32P]-ATP were incubated AMPK for 10 min at 30 °C in the absence or presence of various C2 concentrations. AMP (100 μM) and/or A769662 (20 μM) were included as appropriate positive controls. After 10 min, phosphotransferase activity was quenched by spotting onto P81 ion-exchange chromatography paper  followed by repeated washes in 1% H3PO4 . Statistical tests were conducted using analysis of variance (ANOVA). The values for EC50 and fold stimulation were calculated based on the equation: Activity=Basal+−Basal × [C2])/((EC50)+[C2])). Curve fitting was performed using KaleidaGraph software.AMPK activity assay was conducted as described previouslyPO4 ref. . The levAccession codes: Coordinates and structure factors have been deposited in the protein data bank with the accession codes 4ZHX and 5EZV.How to cite this article: Langendorf, C. G. et al. Structural basis of allosteric and synergistic activation of AMPK by furan-2-phosphonic derivative C2 binding. Nat. Commun. 7:10912 doi: 10.1038/ncomms10912 (2016).Supplementary Figures 1-4, Supplementary Table 1 and Supplementary Notes 1-3.


Hc) is in high demand for densification of magnetic recording. Herein, we report a single-nanosize  hard magnetic ferrite. This magnetic ferrite is composed of ε-Fe2O3, with a sufficiently high Hc value for magnetic recording systems and a remarkably high magnetic anisotropy constant of 7.7 × 106 erg cm−3. For example, 8.2-nm nanoparticles have an Hc value of 5.2 kOe at room temperature. A colloidal solution of these nanoparticles possesses a light orange color due to a wide band gap of 2.9 eV (430 nm), indicating a possibility of transparent magnetic pigments. Additionally, we have observed magnetization-induced second harmonic generation (MSHG). The nonlinear optical-magnetoelectric effect of the present polar magnetic nanocrystal was quite strong. These findings have been demonstrated in a simple iron oxide, which is highly significant from the viewpoints of economic cost and mass production.Development of nanometer-sized magnetic particles exhibiting a large coercive field ( Hc) is the logical next step. In magnetic recording systems910Hc value for writing and reading is ca. 3 kOe. A larger Hc is necessary for future magnetic recording systems, such as bit-patterned media or heat-assisted magnetic recording. Materials exhibiting multiferroic properties are drawing increasing attention12131415162O3 is an attractive material because it exhibits a large Hc value at room temperature718192021222324single-nanosize ε-Fe2O3 spherical particles. The resulting nanomagnets satisfy the required Hc value for magnetic recording applications mentioned above. In addition, they exhibit magnetization-induced second harmonic generation (MSHG), with a strong magnetoelectric (ME) effect. The color of this series is very light, and the absorption coefficient is small in the visible region. In this work, we report the synthesis procedure, the crystal structure, the particle sizes, and magnetic properties of nanometer-size ε-Fe2O3 nanoparticles. Furthermore, we present first-principles calculations for the optical band gap, the spontaneous electric polarization of the polar crystal, and the nonlinear optical-ME effect.Magnetic materials have been used for a whole host of applications: magnetic recording media, permanent magnets, electromagnetic wave absorbers, magnetic fluid, drug delivery, to name a few12345672O3 was prepared from a precursor, in which ferrihydrite Fe10O14(OH)2 nanoparticles were embedded in SiO2 matrix. The details of the synthetic procedure are described in the Methods section. In this report, we describe 18 samples sintered at a large range of temperatures: 250 °C (S-250), 500 °C (S-500), 731 °C (S-731), 902 °C (S-902), 924 °C (S-924), 951 °C (S-951), 979 °C (S-979), 1002 °C (S-1002), 1020 °C (S-1020), 1032 °C (S-1032), 1044 °C (S-1044), 1061 °C (S-1061), 1063 °C (S-1063), 1104 °C (S-1104), 1142 °C (S-1142), 1198 °C (S-1198), 1213 °C (S-1213), and 1295 °C (S-1295).Nanometer-size ε-Fe10O14(OH)2 having hexagonal crystal structure . With increasing sintering temperature, Fe10O14(OH)2 begins to transform into γ-Fe2O3  around 250 °C. As the sintering temperature is raised, γ-Fe2O3 transforms into ε-Fe2O3 around 500 °C. Above 951 °C, the XRPD patterns for S-951–S-1104 show a pure ε-Fe2O3 phase  . In this synthetic method, pure ε-Fe2O3 is obtained in a surprisingly wide sintering temperature range. The range is wider than those of the previously reported methods, including a combination of reverse-micelle and sol–gel methodsX-ray powder diffraction (XRPD) patterns of the sintered samples and the precursor are shown in  S-1020) . Above 1d) of Fe10O14(OH)2 nanoparticles is 2.8 ± 0.5 nm. In the sintering temperature range up to 700 °C, which is the region of the Fe10O14(OH)2 → γ-Fe2O3 transition, the d value is almost constant around 3–4 nm , 6.3 ± 1.7 nm (S-979), 7.8 ± 2.7 nm (S-1002), 8.2 ± 2.7 nm (S-1020), 9.0 ± 2.4 nm (S-1032), 10.5 ± 3.3 nm (S-1044), 11.4 ± 3.8 nm (S-1061), 12.4 ± 3.7 nm (S-1063), and 16.5 ± 4.7 nm (S-1104). The TEM images of all of the samples are shown in The transmission electron microscopy (TEM) image of the precursor shows that the particle size , 0.7 kOe (S-979), 2.1 kOe (S-1002), 3.4 kOe (S-1020), 4.7 kOe (S-1032), 8.3 kOe (S-1044), 11.9 kOe (S-1061), 12.8 kOe (S-1063), 17.3 kOe (S-1104), 20.3 kOe (S-1142), and 20.9 kOe (S-1198)  . The mageference .d dependence of the Hc value. The synthesized ε-Fe2O3 nanoparticles have a size distribution. A theoretical equation for superparamagnetic limit (dp) of nanoparticles with random orientation considering a size distribution was derived by H. PfeifferHc versus d plot was fitted, and the dp value was estimated to be 7.5 nm  a strong magnetic anisotropy due to non-zero orbital angular momentum, L ≠ 0, through a strong hybridization between Fe and O, and (ii) remnants of the magnetic anisotropy due to the polar crystal structure.In addition, we prepared oriented nanocrystal thin film, which was obtained by dispersing the nanocrystals in a vehicle matrix under external magnetic field. The XRPD pattern of the the film . The mag 5.2 kOe , which mS-1020 nanocrystal, which was used for the oriented nanocrystal film mentioned above, is highly transparent, with a light orange color (ε) was only 400 dm−1 cm−1 at 500 nm  program (see Methods). The band structure near the Fermi energy is shown in To understand such a high transparency, the optical band gap of ε-Fe2O3 with Pna21 space group is that of a polar crystal, and therefore, spontaneous electric polarization should be generated. Here, the magnitude of spontaneous electric polarization of ε-Fe2O3 and its origin are investigated by a first-principles calculation. The results show that electric polarization exists along the crystallographic c-axis with a value of 1.0 × 10−1 C m−2, which is large compared with other polar materials2O3 and those of the neutral Fe and O atoms, indicates that the positive charge is distributed on the Fe atoms, and negative charge is distributed on the O atoms, as shown in D site, indicating that the electric polarization along the c-axis at FeDO4 is the main contribution to the pyroelectric property of ε-Fe2O3.The crystal structure of ε-Fe2O3 is considered to be collinear ferrimagnetism18B and FeC are antiparallel to those of FeA and FeD. Based on the molecular-field (MF) theory2O3 can be understood from the product value between the superexchange interaction (J) and the number of exchange pathways (z). The zJ value of the tetrahedral FeD site is smaller than those of octahedral FeA–FeC sites, and hence, thermal fluctuation on FeD sublattice magnetization is larger than FeA–FeC sublattice magnetizations, inducing ferrimagnetism along the crystallographic a-axis at room temperature (The magnetism of ε-Feperature .2O3 has both electric polarization (//c-axis) and magnetic polarization (//a-axis). To investigate the magnetoelectric coupling effect between these two polarizations3031S-1061. When a fundamental femtosecond laser (1064 nm) was put into the sample at 300 K, 532-nm output light was observed (ISH) repeatedly  of 490 K E(ω)E(ω), where PSH and χ(2) are the SH polarization and SH susceptibility tensor, and E(ω) and ω are the electric field and angular frequency of the input fundamental light, respectively (see Methods). The ISH value is related to the crystallographic term TC, ISH is enhanced depending on the magnetization value. From the SHG and MSHG results, the nonlinear optical-ME effect of these nanocrystals is quite strong. That is, the magnetic polarization and electric polarization are strongly correlated to each other in ε-Fe2O3.SH polarization is described by 2O3 magnet. In particular, the Hc value of ε-Fe2O3 with a diameter of 8 nm is 5 kOe, sufficient for magnetic recording systems, where the necessary spec is 3 kOe. Magnetic anisotropy constant approaches 7.7 × 106 erg cm−3, which is over two times as large as those of BaFe12O19 (K = 3.0 × 106 erg cm−3) and SrFe12O19 (K = 3.5 × 106 erg cm−3). The present single-nanosize magnetic material may contribute to the high-density magnetic recording technology, for example, LTO-8 of the next-generation magnetic recording tapes, as well as hard disc drives for computers459102O3 nanomagnet also exhibits spontaneous electric polarization, originating from its polar crystal structure, and an optical-magnetoelectric effect between electric polarization (//c-axis) and magnetic polarization (//a-axis), with an MSHG effect. Surprisingly, this nonlinear optical-ME effect is strong. Importantly, this material possesses high optical transparency with a wide band gap. A transparent magnet will open possibilities for new industrial applications, e.g. transparent electromagnetic wave absorbing windows and magnetic color pigments. Such high performance was achieved in a simple iron oxide, which is significant, since iron oxides are ecofriendly and low cost. Therefore, this material can be reasonably expected to be scalable for industrial applications.In summary, we have developed a synthetic method for a single-nanosize ε-Fe2O3 was prepared from a precursor, where Fe10O14(OH)22. The precursor was sintered at 250 °C–1295 °C for four hours in air, to obtain iron oxide in SiO2 matrix. The SiO2 matrix was then removed by chemical etching using a NaOH aqueous solution.Nanometer-size ε-Feθ–θ scan XRPD measurements were performed using Rigaku Ultima IV and Rigaku RINT2100 with Cu Kα radiation (λ = 1.5418 Å) at 293 K. Rietveld analyses were performed using the PDXL program of RIGAKU. The magnetic properties were measured using a superconducting quantum interference device (SQUID) magnetometer . UV-vis spectra were recorded on JASCO V-670 spectrometer.TEM images were acquired with JEM 2000EX. The 2d value dependence of the Hc value is described byIn the case of nanoparticles with a random orientation considering a size distribution, the VSP and VSW are the mean volumes of superparamagnetic particles and particles with sizes between dp and dp, respectively, and f(d) is the particle size distribution26f(d) in the present analysis.where 2O3 was carried out using the VASP program with the plane-wave projector augmented wave (PAW) method. The spin-polarized density functional theory (DFT) was used as the basis. Approximation of the exchange-correlation functional was done using the generalized gradient approximation (GGA) parameterized by Perdew-Burke-Ernzerhof (PBE).First-principles calculations of ε-FeFor the SHG measurement, incident 1064-nm light was generated by an optical parametric amplifier  pumped by a frequency-doubled Ti:sapphire laser . The incident light was irradiated onto the sample at the angle of 20°. The detection of the reflected SH light (532 nm) was performed by a photomultiplier tube (Hamamatsu R329-02), after passing through color filters.P,iSH = χijk(2)Ej(ω)Ek(ω), where P,iSH and χijk(2) are the SH polarization and SH susceptibility tensor, and the subscripts i, j, and k, refer to the axes of the sample. The space group Pna21 of ε-Fe2O3 has a crystallographic term z-axis (c-axis) and (magnetic polarization) //x-axis (a-axis), the nonzero χijk(2) are TC, the magnetic polarization generates a magnetic term naP21 magnetic space group, that is, χijk(2) are described by the sum of SH polarization is described by How to cite this article: Ohkoshi, S. et al. Nanometer-size hard magnetic ferrite exhibiting high optical-transparency and nonlinear optical-magnetoelectric effect. Sci. Rep.5, 14414; doi: 10.1038/srep14414 (2015).


II atom adopts a distorted square-planar coordination geometry, being C,N-chelated by a 2′,6′-difluoro-2,3′-bipyridine ligand and O,O′-chelated by a pentane-2,4-dionato anionic ligand.The Pt 10H5F2N2)(C5H7O2)], comprises one PtII atom, one 2,6-difluoro-2,3-bipyridine ligand and one acetylacetonate anion. The PtII atom adopts a distorted square-planar coordination geometry, being C,N-chelated by the 2,6-difluoro-3-(pyridin-2-yl)pyridin-4-yl ligand and O,O′-chelated by the pentane-2,4-dionate ligand. The two pyridine rings of the bipyridine ligand are approximately coplanar, making a dihedral angle of 1.2 (2)°. A variety of intra- and intermolecular C—H⋯O and C—H⋯F hydrogen bonds, as well as π–π interactions [centroid–centroid distances = 4.337 (3) and 3.774 (3) Å] contribute to the stabilization of the molecular and crystal structures, and result in the formation of a three-dimensional supramolecular framework.The asymmetric unit of the title compound, [Pt(C C,N-chelating ligands have been considered as an attractive research area due to their wide applications, such as biological imaging, non-linear optics, oxygen sensing and organic light-emitting diodes (OLEDs)  compound with fluorinated bipyridine and acetylacetonate  ligands.Cyclometalated platinum(II) compounds with II atom, one 2,6-difluoro-2,3-bipyridine ligand and one acetylacetonate anion. The PtII atom is four-coordinated by the C,N-chelating 2′,6′-difluoro-2,3′-bipyridinato ligand and by the O,O′-chelating pentane-2,4-dionato ligand, forming a distorted square-planar coordination sphere due to narrow ligand bite angles, which range from 81.28 (17) to 93.25 (13)°. The Pt—C bond length of 1.951 (4) Å is shorter than the Pt—N bond length of 1.995 (4) Å due to the more electronegative fluorine substituent on the C-bound pyridine ring. The Pt—C, Pt—N and Pt—O bond lengths (acac)] °, indicating that an effective π conjugation of the two pyridine rings occurs in the title compound. The molecular structure is stabilized by weak intramolecular C—H⋯O and C—H⋯F hydrogen bonds  Å and Cg1—Cg2ii = 3.774 (3) Å, where Cg1 and Cg2 are the centroids of the N1, C1–C5 and the N2, C6–C10 rings, respectively; symmetry codes: (i) −x + 1, −y + 2, −z + 2; (ii) −x + 2, −y + 2, −z + 2], resulting in the formation of an overall three-dimensional supramolecular framework  = 0.95 Å, Uiso(H) = 1.2Ueq(C) for Csp2-H, and 0.98 Å, Uiso(H) = 1.5Ueq(C) for methyl protons.Crystal data, data collection and crystal structure refinement details are summarized in Table 310.1107/S2056989015004375/wm5128sup1.cifCrystal structure: contains datablock(s) I, New_Global_Publ_Block. DOI: 10.1107/S2056989015004375/wm5128Isup2.hklStructure factors: contains datablock(s) I. DOI: 1051825CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: