This study separated and purified the β-mannanase from Bacillus subtilis BE-91, which is a powerful hemicellulose-degrading bacterium using a “two-step” method comprising ultrafiltration and gel chromatography. The purified β-mannanase (about 28.2 kDa) showed high specific activity . The optimum temperature and pH were 65°C and 6.0, respectively. Moreover, the enzyme was highly stable at temperatures up to 70°C and pH 4.5–7.0. The β-mannanase activity was significantly enhanced in the presence of Mn2+, Cu2+, Zn2+, Ca2+, Mg2+, and Al3+ and strongly inhibited by Ba2+ and Pb2+. Km and Vmax values for locust bean gum were 7.14 mg/mL and 107.5 μmol/min/mL versus 1.749 mg/mL and 33.45 µmol/min/mL for Konjac glucomannan, respectively. Therefore, β-mannanase purified by this work shows stability at high temperatures and in weakly acidic or neutral environments. Based on such data, the β-mannanase will have potential applications as a dietary supplement in treatment of inflammatory processes. Konjac glucomannan is a randomly arranged polymer of β-1,4-linked glucose and mannose residues at ratio of 1.0 : 1.6. Both the backbones of mannan and Konjac are modified by α-1,6-linked galactosyl residues to form galactomannan and galactoglucomannan, respectively [Mannan consists of a series of complex polysaccharides, which are found in the cell wall of marine algae . The bacectively .β-mannanase (EC 3.2.1.78) is a hemicellulase that attacks the internal glycosidic bonds of mannan backbone to release the condensed β-1,4-manno-oligosaccharides [β-mannanases are widely applied in pulp and paper processing [β-1,4 mannopyranoside linkage in mannan, galactomannan, glucomannan, and galactoglucomannan.anase EC .2.1.78 iocessing , food [6β-mannanase is widely produced by bacteria [β-mannanase from bacteria is wildely used because of numerous advantages, including extracellular secretion, economic production and purification, and novel characteristics, such as tolerance to heat and alkaline conditions [bacteria , 10, actbacteria , fungi [bacteria , plants,bacteria . Among tnditions .β-mannanase-producing bacteria have been reported [β-mannanase has been proposed to meet the industrial demands [β-mannanase with lower energy for production has attracted considerable interest over the past few years; however, it has rarely been characterized. It is clarified that β-mannanase with high activity in short fermentation time confers lower costs during the production procedures. Therefore, the exploitation of strains producing high β-mannanases activity is valuable and profitable. In current study, we isolated and preserved a powerful hemicellulose-degrading bacterium (BE-91). Then we explored the efficient purification process and characterized the enzymatic properties of its β-mannanase.Although multiple reported , 16, the demands . HoweverB. subtilis BE-91, a strain used for herbaceous fiber extraction, was identified and preserved by the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Science . B. subtilis BE-91 was cultured in Petri dish containing 0.5% yeast extract, 1% NaCl, 0.5% Konjac glucomannan, 1% bacto tryptone, 0.05% trypan blue, and 1.5% agar. The seed medium was mainly composed of 0.1% glucose, 0.4% Konjac glucomannan, 0.3% beef extract, 0.2% yeast extract, 0.5% peptone, and 0.5% NaCl. The fermentation medium primarily consisted of 0.2% yeast extract, 0.7% Konjac glucomannan, 0.5% peptone, 0.3% beef extract, and 0.5% NaCl. B. subtilis BE-91 was first activated in the seed medium at 35 ± 1°C for 5.5 h. Subsequently, the suspension was serially diluted, spread onto Petri dishes, and incubated at 35 ± 1°C for 18 h. The single colony exhibiting the largest hydrolytic halo was transferred into an Erlenmeyer flask with the seed medium and cultured at 35 ± 1°C for 6 h at 180 rpm. Consequently, 2% culture was inoculated in the fermentation medium and cultured for 6 h at 35 ± 1°C at 180 rpm [The 16S rDNA of strain BE-91 was PCR amplified from genomic DNA using the Bacterial Identification PCR Kit . The obtained 16S rDNA was sequenced by the ABI 3730XL 96-capillary DNA analyzer. The primers were as follows: P1 5′-AGAGTTTGATCMTGGCTCAG-3′ and P2 5′-TACGGYTACCTTGTTACGACTT-3′. The resulting sequence aligned closely with the related standard sequences of other bacteria retrieved from GenBank. Neighbor-joining clusters were constructed by Mega 6.0 .β-mannanase activity was estimated by initiating the reaction at 65°C for 10 min in 0.05 mol/L citric acid/0.1 mol/L Na2HPO4 buffer (pH 6.0) with 0.2% (w/v) Konjac glucomannan as substrate. The amounts of reducing sugar in the reaction were quantified based on a standard curve generated with mannose using the 3,5-dinitrosalicylic acid (DNS) method. One unit (IU) of β-mannanase activity was defined as the amount of protein producing 1 μmol/L of reducing sugar per minute  under standard conditions [nditions .β-mannanase was purified using a two-step process involving ultrafiltration  and gel filtration. The fermentation liquid was fractionated orderly by 100 kDa, 50 kDa, and 5 kDa membrane thresholds. The solution filtered with 5 kDa < MW < 50 kDa was further purified on a Sephadex G-100 gel column . The eluate was obtained at a rate of 0.5 mL/min and collected in 5 mL fractions. β-mannanase activity was determined by the DNS method, whereas the protein was quantified by the Coomassie brilliant blue staining against bovine serum albumin (BSA) standard [The bacterial standard .β-mannanase was determined by SDS-PAGE , with 3% stacking gel and 12% separating gel [β-mannanase was derived from the relative mobility of molecular weight markers resolved simultaneously.The molecular mass of the ting gel . The proting gel . The molβ-mannanase was assayed at a range of temperatures between 50 and 70°C in 0.05 mol/L citric acid-0.1 mol/L Na2HPO4 buffer at pH 6.0. The thermostability was assessed by preincubating the enzyme, without a substrate, at different temperatures varying over 20–80°C for 30 min. The residual activity was promptly measured by the DNS method. The β-mannanase activity was considered to be 100% when preincubated at 4°C.The activity of β-mannanase activity was evaluated by incubating the purified enzyme at different pH conditions ranging from 4.0 to 8.0 in 0.05 mol/L citric acid-0.1 mol/L Na2HPO4 buffer at 4°C. The stability at a particular pH was tested by preincubating the purified enzyme, without a substrate, for 30 min in various 0.05 mol/L citric acid-0.1 mol/L Na2HPO4 buffers at pH 3.0–8.5 at 4°C. The residual β-mannanase activity was immediately measured after treatment by the DNS procedure.β-mannanase, the enzyme was incubated for 30 min at 4°C in the presence of various 1.0 mmol/L metal ions, CaCl2·2H2O, ZnCl2, FeCl3, PbCl2·2H2O, MnCl2·4H2O, MgCl2·6H2O, KCl, CuCl2·2H2O, AlCl3, BaCl2, and NH4Cl. The residual β-mannanase activity was measured at a specific condition and that of the treatment in the absence of additives as a control.In order to examine the effects of metal ions on the activity of  Ceatonia siliqua seeds , carob galactomannan , guar galactomannan , ivory nut mannan , 1,4-beta-D-mannan , wheat arabinoxylan , beechwood xylan , and carboxymethyl cellulose  were examined. In brief, 0.2% (w/v) glycans were incubated with β-mannanase at 65°C for 10 min in 0.05 mol/L citric acid-0.1 mol/L Na2HPO4 buffer at pH 6.0, and the reducing sugars were measured by DNS. The Michaelis-Menten kinetic parameters, Vmax and Km, were calculated for β-mannanase. The assays of the purified enzyme were carried out by the standard DNS procedure, as described above, using 1–5 mg/mL locust bean gum and 0.5–2.5 mg/mL Konjac glucomannan as substrates. The kinetic constants were determined from the Michaelis-Menten equation by directly inputting the initial rates from Lineweaver-Burk plots or the nonlinear regression [Various glycans, such as Konjac glucomannan , locust gression .β-mannanase activity experiment was performed in triplicate and expressed as mean ± SD (standard deviation). The statistical analyses were performed with SPSS 15.0 . One-way or two-way analysis of variance (ANOVA) was used to compare various treatment groups.Each β-mannanase activity assay. β-mannanase activity of the four bacteria (strain BE-23 without β-mannanase activity was used as a negative control). Strain BE-91 fermented for 9 h exhibited the highest activity, up to 273.7 IU/mL. Wild-type B. subtilis MA139 yielded a maximum β-mannanase activity of 170 IU/mL after 3 days of fermentation, and the maximum enzyme activity of B. subtilis TJ-102 was 205.3 IU/mL at 38 h [β-mannanase with higher activity in shorter time.Four bacteria were stochastically selected for the  at 38 h , 26. Not B. subtilis 16S rDNA (gi 530330588 and gi 341831474) was 99%. It was confirmed that the similarity of B. subtilis type strains about 16S rRNA gene sequence is higher than 98% [ B. subtilis isolates.The 1,508 bp sequence of 16S rDNA of strain BE-91 was analyzed by a phylogenic tree . The homthan 98% , 28. We β-mannanase in B. subtilis BE-91 exceeded 66.0%; multiple purifications achieved 32.9-fold pure β-mannanase activity, and the specific activity of the purified enzyme reached 79,859.2 IU/mg. The purified β-mannanase was shown to be homogeneous judged by SDS-PAGE analysis  [β-mannanases from Penicillium occitanis Po16 and Bacillus halodurans PPKS-2 were 22 and 18 kDa, respectively [The apparent molecular weight of 28.2 kDa , lower t 32 kDa) –34. Simiectively , 31. Dueectively .β-mannanase was maximally active at 65°C  [β-mannanase of BE-91 showed a pronounced activity at higher temperatures. As compared to the thermostability of the β-mannanase from wild-type B. subtilis BCC41051 (60°C for 30 min) [β-mannanase retains 80% residual activity after incubation at 20–70°C for 30 min, indicating enhanced thermostability.The purified  at 65°C  and rema at 65°C . Comparesp. DZ3) , 31, 36, 30 min) , this β-β-mannanase were measured at various pHs. The optimum enzyme activity was obtained at pH 6.0 (β-mannanase was the same as that of B. subtilis MA139 (pH 6.0), an enzyme that can potentially be used as a feed additive for monogastric animals [β-mannanase activity was negligible, retaining <80% of its maximal value obtained after incubation at pH > 7.5, 4°C for 30 min. A relatively broad zone of optimum activity was observed. Therefore, BE-91 β-mannanase can be considered a weakly acidic and neutral enzyme, thereby rendering suitability for animal feed industry [The optimal pH and the stability of BE-91 t pH 6.0 , and mort pH 6.0 . Interes animals . At pH <industry .β-mannanase activity was measured  than the locust bean gum , similar to the values obtained for Penicillium pinophilum C1 and Penicillium freii F63, hence constituting it as an adequate candidate in food industry for the production of oligosaccharides [charides , 18, 39.B. subtilis bacteria are abundant, moderately stable, and mostly nonpathogenic microorganisms. Our results indicated that B. subtilis BE-91 could be considered a prominent candidate for the production of extracellular β-mannanase. In addition, this study developed an advanced purification approach, “two-step method,” with high efficiency, high yield, and easy operation. Furthermore, the β-mannanase purified from BE-91 was extremely stable at relatively high temperatures and various weak acidic or neutral pHs. Finally, the enzyme showed a higher affinity towards natural Konjac glucomannan, a major functional food material. Therefore, this β-mannanase, purified and characterized from B. subtilis BE-91 for the first time, is suitable for inflammatory diseases.


The crystal structure revels chelation of ligand through selenium donors with a natural bite-angle of 110.54 (1)°A palladium(II) complex containing a bis(selenium) ligand based on cyclodiphosph(V)azane,  tert-butyl-2,4-bis(tert-butylamino)-1,3,2λ5,4λ5-diazadiphosphetidine-2,4-diselone-κ2Se,Se′]palladium(II)}, cis-[PdCl2{I}], (II), containing a bis(selenium) ligand based on cyclodiphosph(V)azane, cis-[(tBuNH)(Se)P(μ-NtBu)2P(Se)(NHtBu)], (I), has been synthesized and structurally characterized. The crystal structure of complex II reveals that the ligand chelates through selenium donors with the natural bite-angle of 110.54 (1)° and a Pd—Se bond distance of 2.444 (1) Å. The coordination around PdII shows a slightly distorted square-planar geometry, as indicated by the angle between the [PdCl2] and [PdSe2] planes of 5.92 (3)°. In the crystal, the molecules are interlinked through weak N—H⋯Cl and C—H⋯Cl hydrogen-bonding interactions.A palladium(II) complex {systematic name: dichlorido[1,3-di- III–N ring systems with general formula, cis-[RP(μ-NtBu)2PR]. The planar nature of the four-membered ring favors a bridging bidentate coordination mode through phosphorus donors rather than chelation, to afford structurally interesting macrocyclic and polymeric complexes azanes, cis-[R(E)P(μ-NtBu)2P(E)R]  and its amide derivatives has been studied extensively by Stahl  have been developed over the years and have shown promising catalytic activity for several reactions  with a wide bite-angle selenium ligand based on cyclodiphosph(V)azane cis-[(tBuNH)(Se)P(μ-NtBu)2P(Se)(NHtBu)], (I).Cyclodiphosph(III)azanes are four-membered Ptahl 2000 and BriaII complex (II) is presented in Fig. 1II confirms the chelation of cis-[(tBuNH)(Se)P(μ-NtBu)2P(Se)(NHtBu)] (I) through selenium donors to the [PdCl2] moiety, with a Se1—Pd1—Se2 natural bite-angle of 110.54 (1)°. The rigid four-membered cyclodiphosphazane [P(μ-NtBu)2P] ligand backbone enforces large natural bite-angles. The Se1—Pd1—Cl1 and Se2—Pd1—Cl2 bond angles are 79.27 (2) and 79.36 (2)°, respectively, smaller than the natural square-planar angle, whereas the Cl1—Pd1—Cl2 angle [91.19 (2)°] is closer to the typical value for a square-planar angle. In complex II, the exocyclic Se1—P1—N3 and Se2—P2—N4 angles at 114.32 (7) and 117.13 (7)°, respectively, are slightly larger than the corresponding angle in the uncoordinated ligand I  in the uncoordinated ligand (I). This may be a result of the coordination of Se to the Pd center. The Pd—Cl bond distances [Pd1—Cl1 = 2.3381 (6) and Pd1—Cl2 = 2.3159 (6) Å] are consistent with those reported for PdII complexes with Se donor ligands 2P] ring in complex II is greatly puckered, as indicated by the angle of 22.61 (2)° between the N1/P1/N2 and N1/P2/N2 planes. The corresponding dihedral angle for the uncoordinated ligand is 3.73 (2)°.A perspective view of the molecular structure of the PdII, the exocyclic nitrogen substitutents are arranged in an endo, endo fashion, whereas in ligand I they are arranged in exo, endo orientations (Se)P(μ-tBuN)2P(Se)(NHtBu)], (I), was prepared following a reported procedure  was added dropwise to a solution of cis-[(tBuHN)(Se)P(μ-tBuN)2P(Se)(NHtBu)]  in 10 mL of CH2Cl2 under an N2 atmosphere at ambient temperature. The resultant dark-orange solution was stirred for 6 h. The solution was then concentrated to 10 mL, diluted with 10 mL of pentane, and stored at 248 K for a day to afford the analytically pure orange crystalline product. X-ray quality crystals were obtained by slow evaporation from a DMF solution at room temperature. Yield: 76% , m.p. 455–457 K.A dichloromethane solution (10 mL) of : 247 (12068), 294 (15752), 355 (6827). Analysis calculated for C16H38N4P2Se2PdCl2: C, 28.11; H, 5.60; N, 8.19. Found: C, 28.37; H, 6.01; N, 28.74.Uiso(H) = 1.2-1.5 times those of the parent atoms. The t-butyl group attached to N4 was modeled as rotationally disordered over two sites of approximately equal population. These were refined with restraints so that the geometries of the two components of the disorder are comparable.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018000841/nk2239sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989018000841/nk2239Isup2.hklStructure factors: contains datablock(s) I. DOI: 1549758CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Antidiabetic activity of compounds 1–5 in terms of their cellular antioxidant and free radical scavenging and also in streptozotocin- (STZ-) induced diabetic mice was evaluated on liver transaminases, lipid peroxidation, total bilirubin, total protein, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (CSH-Px), and glutathione reductase (GSH). Results indicated that 1–5 scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (∙OH), nitric oxide radicals (NO∙), superoxide anion radical (O2∙−), radical cation (ABTS∙+), and hydrogen peroxide (H2O2) radical, and protection against H2O2 induced BSA damage was also observed. Furthermore, 1–5 showed ability to decrease the oxidative stress in H9c2 cell. Diabetic mice present high levels of lipid peroxide, total protein, SGPT, SGOT, ALP, and TB. However, treatment of STZ-induced diabetes in mice with 1–5 reduced levels of these enzymes leading to protector effect of liver. In addition, with treatment with 1–5, increases in radical scavenging enzymes of CSH-Px, SOD, GSH, and CAT have also been observed in diabetic mice. The antioxidant properties of compounds 1–5 are a promising strategy for ameliorating therapeutic effects by avoiding disorders in the normal redox reactions in healthy cells which consequently could alleviate complications of diabetes.Six new flavonoids 2′,4′-dihydroxychalcone-6′-O- ROS can be responsible for the attack to biological macromolecules such as nucleic acids, proteins, membrane lipids, and carbohydrates, causing damage in the cell, which has been implicated in cardiovascular diseases, cancer, neurodegenerative disorders, and diabetes [Oxidative stress is a consequence of the increased reactive oxygen species (ROS) production and/or a decrease in their elimination. ROS such as hydrogen peroxide  Sarg, belonging to the Leguminosae family, is known as “palo azul” and has widely been used as antirheumatic, for the treatment of nephrolithiasis and bladder disorders developed in diabetes [ E. polystachya contains polyphenols [ E. polystachya was evaluated in in vitro assays, showing antioxidant potential, hypoglycemic, and AGEs inhibition capacity [ E. polystachya in in vitro and in vivo assays.The treediabetes . Phytochyphenols . In anotyphenols . In prevcapacity . In thislH-lH, l3C, DEPT, ID, DQF-COSY, TOCSY, and HMBC experiments were recorded on a Bruker DRX-300 NMR spectrometer, operating at 599.19 MHz for 1H and 150.86 MHz for l3C, using the UXNMR software package; chemical shifts are expressed in δ (ppm) using TMS as an internal standard [IR spectra were determined on a Perkin-Elmer 1720 FTIR.  E. polystachya was collected in October 2014 near Tula, State of Mexico. A voucher specimen (number 49584) is deposited at the Herbarium of Universidad Autonoma Metropolitana-Xochimilco, Mexico.Bark of E. polystachya (5 kg) was milled and macerated with distilled water and methanol (1 : 1) for 15 days. The extract was evaporated under reduced pressure at 40°C, affording 400 g which was partitioned sequentially with n-hexane, CHCl3, and MeOH. The methanol extract was adsorbed on 50 g of silica gel 60 (70−230-mesh ASTM), loaded onto a column of silica gel of 50 cm length and 7 cm diameter, 300 g silica gel, and then eluted with CH2Cl2/EtOAc 9 : 2 to yield 9 subfractions (PA-1 to PA-9). The elutions were examined by TLC and each one of them was determined as antioxidant activity. The fractions having antioxidant activity were PA-5 and PA-7. PA-5 was subjected to silica gel column chromatography eluted with EtOAc/hexane 7 : 2 to yield 7 subfractions (PA5-1 to PA5-7). Subfractions PA5-2 and PA5-7 were subjected to gel filtration over Sephadex LH-20 eluted with CHCl3/MeOH (6 : 1), and this led to the isolation of compounds 7–13 which were combined and further purified by repeated preparative thin layer chromatography (PTLC) eluting with ethyl ether/acetone/methanol (1.5/3/0.5) gradient system. This led to the isolation of compounds 7 (65.0 mg), 8 (13.5 mg), 9 (76.4 mg), 10 (15.8 mg), 11 (49.2 mg), 12 (29.37 mg), and 13 (48.5 mg). PA-7 was subjected to silica gel column chromatography eluted with EtOAc/hexane 9 : 3 to yield 6 subfractions (PA7-1 to PA7-6). Fractions PA7-4 and PA7-5 were combined and further crude mixture was subjected to preparative thin layer chromatography (PTLC) eluting with ethyl ether/acetone/methanol (4/2/1.0) gradient system. Subfractions 1–3 were purified by gel filtration over Sephadex LH-20 eluted with CH2Cl2-CH3OH 1 : 1 increasing concentration of MeOH yielding compounds 1 (67.1 mg), 2 (85.4 mg), 3 (72.8 mg), 4 (96.7 mg), 5 (90.2 mg), and 6 (42.9 mg).The dried bark 1 mL solution of 2,2-diphenyl-1-picrylhydrazyl  in methanol was added to 4 mL of sample. The mixture was shaken vigorously and was allowed to stand at room temperature for 30 min. Absorbance of the resulting solution was measured at 517 nm using a spectrophotometer  [μmol/L FeCl2 was reacted with flavonoids for 5 min. Then ferrozine (200 μmol/L) was added and the mixture was left to stand for another 10 min. The absorbance at 562 nm was determined spectrophotometrically  [40 2O2 alone in 30 mmol/L acetate buffer with pH 3.6 was incubated for 30 min at room temperature producing 10 mmol/L of ABTS radical cation (ABTS∙+). 80 μL of this solution was added to 0.4 mol/L acetate buffer  and to 20 μL of the sample solutions; after exactly 5 min the absorbance was read at 734 nm. A dose-response curve was plotted for Trolox and antioxidant ability was expressed as TEAC value [ 2 mmol/L HAC value .μg/mL of the sample; then the tubes were incubated for 150 min at room temperature. Griess reagent (0.3 mL of 1 g/L naphthylethyl-enediamine dihydrochloride and 0.3 mL of 10 g/L sulfanilamide in 5% H3PO4) was added to the incubation solution. The absorbance was immediately read at 570 nm and referred to the absorbance of standard solutions of sodium nitrite salt treated in the same way with Griess reagent [In the test tubes to 0.1 mL of sodium nitroprusside solution (25 mmol/L) was added 150 2O2 (20 mmol/L), and 0.4 mL of 20 mmol/L phosphate buffer (pH 7.4), for 2 h at 37°C. 5,5-Dithio-bis(2-nitrobenzoic acid)  was then added and the mixture was left to stand for another 30 min. The absorbance was measured at 410 nm. The free thiol concentration of samples was calculated based on the standard curve prepared by using various concentrations of l-cysteine. 0.2 mL of samples was incubated with 1 mL BSA (40 mg/mL), 0.4 mL HμM riboflavin, 58 mM phosphate buffer at pH 7.6, and 50 μM of nitroblue tetrazolium chloride (NBT). The reaction mixture is exposed for 15 min to 40 V under fluorescence lamp to initiate the reaction. The absorbance was determined at 560 nm [The reaction mixture is carried out in final volume of 3 mL containing 6 mM EDTA, 20 t 560 nm .μM), hydrogen peroxide (500 μM), ascorbic acid (100 μM), and EDTA (100 μM) and test sample (10–1000 μg/mL−1) in a final volume of 1 mL. The mixture was incubated at 37°C for 1 h. Then 0.8 mL of the mixture was added to 2.8% of trichloroacetic acid (TCA) solution (1.5 mL), followed by sodium dodecyl sulphate (0.2 mL) and a thiobarbituric acid (TBA) solution (1 mL of 50 mM at 1% in sodium hydroxide). The mixture was heated (90°C for 20 min) to develop the colour. After cooling, the absorbance was determined at 532 nm [The reaction mixture contained phosphate buffer , 2-deoxy-2-ribose (2.8 mM), ferric chloride  solution and then were mixed with 600 mixtures .2 and 95% air at 37°C, after the cells were plated in 35-mm culture dishes at 5.0 × 10−4 cells/cm2. Then, the medium is changed with a new one. Cells seeded on 96-well plates were incubated with DCFHDA probe for 40 min. At the end of this period, medium was removed and cells were exposed to the flavonoids under investigation at a concentration of 10, 50, and 100 μg/mL. After incubating exposed cells at 37°C for 24 h, fluorescence was measured at 488 nm (excitation) and 535 nm (emission) wavelengths on a microplate reader (Molecular Devices Spectra MAX Gemini X).The intracellular accumulation of ROS in the H9c2 rat cardiomyoblast cell line was determined with carboxy-2′,7′-dichloro-dihydro-fluorescein diacetate (DCFHDA) method . Cells HThe study was conducted in male CD1 mice, weighing about 25–30 g. Before and during the experiment, animals were fed a standard laboratory diet  with free access to water. Mice were procured from the bioterium of ENCB and were housed in microloan boxes in a controlled environment (temperature 25 ± 2°C). Animals were acclimatized for a period of three days in their new environment before the initiation of experiment. Litter in cages was renewed three times a week to ensure hygiene and maximum comfort for animals. The experiments reported in this study were following the guidelines stated in Principles of Laboratory Animal Care  and the Mexican Official Normativity (NOM-062-Z00-1999). All animals procedures were performed in accordance with the recommendations for the care and use of laboratory animals (756/lab/ENCB).Mild diabetes type 2 was induced in overnight fasted mice by a single intraperitoneal injection of 45 mg/kg streptozotocin (STZ) dissolved in 0.1 mol/L cold citrate buffer (pH 4.5), 15 min after the intraperitoneal administration of 120 mg/kg nicotinamide. The STZ treated animals were allowed to drink 5% glucose solution overnight to overcome drug induced hypoglycemia. After 7 days of development of diabetes, mice with moderate diabetes having persistent hyperglycaemia with more than 200 mg/dL were used for further experimentation [n = 10) of diabetic mice were used to determine the chronic effect of 1–5. Each group was submitted to a specific treatment, as follows. Normal control and mild diabetic rats groups were fed with normal diet and drinking water ad libitum and were given saline by gastric gavage Mice with mild diabetes daily treated with the compounds isolated by an oral gavage at doses of 20 mg/kg of weight. The flavonoids were dissolved in distilled water and administered orally as a daily dose for four weeks. Mice were fasted overnight and sacrificed by cervical dislocation. Blood samples were collected in EDTA coated tubes. Livers, kidney, and pancreas were removed, washed with cold saline, and stored at −80°C.Eight groups  as thiobarbituric acid reactive substance was measured at 532 nm spectrophotometrically [Activities of superoxide dismutase (SOD), serum catalase (CAT), glutathione peroxidase (CSH-Px), and glutathione reductase (GSH) were measured by assay kits purchased from Cayman Chemical , and the procedures were according to the kits instructions. The protein concentration was determined by the Bradford method  as descrtrically . Serum g 1–13  are presented as follows.The MeOH extract was subjected to multiple chromatographic purifications to afford dihydrochalconesβ-d-glucopyranoside (1) was isolated as dark yellow amorphous powder, mp 138–140°C; UV λmax 362, 301, and 238 nm; IR (KBr) cm−1: 3341, 2957, 2761, 1611, 1597, 1506, and 1463; and HRESIMS m/z 418.1270 calc. For C21H22O9, 418.1264, and 1H and 13C NMR data see 2′,4′-Dihydroxychalcone-6′-O- 1 was isolated from ethanol-water 1 : 1 extract of bark of Eysenhardtia polystachya after purification through silica gel column chromatography. Compound 1 showed a molecular ion at m/z 418.1270 in its HREIMS and the presence of 21 carbons on the 13C NMR spectrum suggested a molecular formula C21H22O9. DEPT experiments classified the protonated carbon signals into one methylene, fourteen methines, and six quaternary carbons. The IR spectrum of 1 showed absorption bands at νmax 3341, 2957, 2761, 1597, 1506, 1463, and 1611 cm−1 showing the presence of hydroxy, aromatic, and carbonyl groups. The 1H NMR spectrum showed the presence of unsubstituted ring B , and it showed a pair of doublets of δH 8.24 (H-β) and δH 7.50 (H-α) with the vicinal coupling constant 3J = 15.8 Hz due to trans-α,β-unsaturated ketone protons. Its ketone carbonyl was confirmed with the signal at δC 202.69, C-α (δC 141.31), and C-β (δC 130.60) in the 13C NMR being indicative of chalcone skeleton. NMR spectrum showed signals for one chelated hydroxyl group δH 13.2  and one singlet at δH 6.81 assigned to OH-4′. In addition 1H NMR spectrum showed the presence of two aromatic protons in ring A (δC 103.79) and C-4′ (δC 148.36), H-5′ with C-1′ (δC 103.79), C-3′ (δC 114.82), C-4′ (δC 148.36), and C-6′ (δC 161.21), and H-1′′ with C-6′ (δC 130.60), C-2′′ (δC 78.36), and C-5′′ (δC 71.17) was observed. Thus, the structure of compound 1 was established as 2′,4′-dihydroxychalcone-6′-O-β-d-glucopyranoside  was isolated as dark yellow amorphous powder, mp 150–152°C; UV λmax 235, 264, 302, and 312 nm; IR (KBr) cm−1: 3339, 1621, 1598, 1510, and 1460; and HRESIMS m/z 644.1936 calc. For C28H36O17, 644.1952, and 1H and 13C NMR data see  2 was also isolated as dark yellow amorphous powder. The aliphatic hydroxyl group of compound 3 has α position similarly to 2. The singlet at δH 3.85 was assigned to an aromatic methoxyl group which showed long range correlation with C-4 (δC 145.05) in the HMBC spectrum indicated to be linked at position C-4, whereas the doublets at δH 4.85 and δH 5.01 were attributed to two β-glucopyranose moieties on the basis of the coupling constant (7.5 and 7.3 Hz). In the HMBC spectrum of 2, the long range correlations of anomeric proton H-1′′ (δH 4.85) with C-2′ (δc 164.13), C-3′ (δc 107.88), C-4′ (δc 165.34), C-2′′ (δc 74.73), C-3′′ (δc 78.85), and C-5′′ (δc 77.32) were observed. These signals suggested that glucose unit should be directly linked at position C-3′. Also the anomeric proton H-1′′′ (δH 5.01) correlation with C-6′ (δc 164.04), C-2′′′ (δδC 78.65), and C-5′′′ (δc 78.01) indicated that the other glucose unit should be linked to C-6′ through a glycosidic bond -1-benzopyran-2-one (3) was isolated as dark yellow amorphous powder, mp 142–144°C; UV λmax 249 and 381 nm; IR (KBr) cm−1: 3439, 1659, 1619, 1521, and 1443; HRESIMS m/z 514.1483 calc. For C26H26O11, 514.1475, and 1H and 13C NMR data see 7-Hydroxy-5,8′-dimethoxy-6′ 3 was isolated as dark yellow amorphous powder. Its molecular formula was assigned as C26H26O11 on the basis of HRESIMS and 13C NMR data. The IR spectrum showed characteristic signals for OH (3439 cm−1), aromatic ring (1521 and 1443 cm−1), and α,β-unsaturated ketone (1659 and 1619 cm−1). The UV spectrum at 249 and 381 nm confirmed the existence of unsaturated functional groups. The 1H NMR spectrum showed one single proton at δH 14.15  and two methoxyl groups at δH 3.39  and δH 3.46 , while the resonances of two trans-coupled olefinic protons were recorded at δH 7.32  and 7.32  as well as two olefinic protons δH 6.58  and δH 7.50 . In addition 1H NMR spectrum showed aromatic signals of 4H at 6.32–6.47 and also exhibited signals due to α-l-rhamnopyranosyl at δH 5.05  and δH 1.15 . 13C NMR and DEPT spectra  showed long range correlation with C-6′ at δC 163.56. 13C NMR data, with the corresponding literature, suggested that 3 exhibited the skeletons of a neoflavone and a chalcone [δH 6.58 with C-2 (δC 168.21) and C-4a (δC 105.97) and H-4 with C-3 (δC 143.48) revealed the existence of the neoflavone skeleton. The HMBC relationship of the double bond group (δH 7.97 and δH 7.70) indicated a fragment of O@CAC@CAC, showing the skeleton of a chalcone. Compound 3 possesses a methoxyl group at C-5 suggested by a 13C-1H long range coupling from OCH3-5 (δH 3.39) to C-5 (δC 143.48) and another methoxyl group in C-8′ indicated by correlations observed in OCH3-8′ (δH 3.46) to C-8′ (δC 166.12) in the HMBC spectrum (α-l-rhamnopyranosyl-8-(3-phenyl-trans-acryloyl)-1-benzopyran-2-one  was isolated as yellow amorphous powder, mp 156–158°C; UV λmax 228 and 349 nm; and HRESIMS m/z 396.0863 . For 1H and 13C NMR data, see 6′7-Dihydroxy-5,8-dimethoxy-8(3-phenyl-trans-acryloyl)-1-benzopyran-2-one  was isolated as dark yellow amorphous powder, mp 142–134°C; UV λmax 212, 292, and 344 nm; IR (KBr) cm−1: 3389, 1619, 1547, 1483, and 1429; and HRESIMS m/z 368.1641 calc. For C22H24O5, 368.1624, and 1H and 13C NMR data see 9-Hydroxy-3,8-dimethoxy-4-prenylpterocarpan , δH 3.59, , δH 3.50 , and δH 5.20  related to the protons of the heterocyclic ring B. The HMQC spectrum suggested the presence of two OCH3 groups at δH 3.11  and δH 3.35 , confirming its presence in the 13C NMR with signals to δC 55.57 (OMe-3) and δC 60.64 (OMe-8). HMBC spectrum shows the relations between 3.11  and δC 147.76  and between 3.77  and δC 152.51  show two -OMe that joined with C-3 and C-8 and the relations between OH δH 8.12  and δC 99.01 (C-10) and between δC 163.17  and δC 152.51  indicate that hydroxyl group is at C-9. The proton doublets at δH 7.15 and δH 6.52 suggested ortho-coupled position. The correlation of H-1′ (δH 3.43) with C-4 (δC 115.84), C-4a (δC 154.59), and C-3 (δC 147.76) in HMBC spectrum indicated that prenyl group was located at C-4 of A ring. The HMBC spectra (δH 5.20) with C-1 (δC 77.75), C-6a (δC 40.48), and C-11b (δC 77.75) and H-6a (δH 3.50) with C-6a (δC 69.37), C-11a (δC 77.75), and C-6b (δC 116.56) (δH 5.20) with H-1 (δH 7.15) suggested that H-11a is equatorially oriented cis-ring fusion between rings B and C. The cisconfiguration of 6a and 11a was confirmed based on the evidence from the chemical shift and J value (7.0 Hz) compared with values reported in the literature for cis (6.6 Hz) and trans (13.4) [ 5 are shown in  6 is 9-hydroxy-3,8-dimethoxy-4-prenylpterocarpan (Compound spectra  show cor 116.56) . NOESY cs (13.4) . Key corrocarpan .α,4,4′-Trihydroxydihydrochalcone-2′-O-β-d-glucopyranoside (6) was isolated as dark yellow amorphous powder, mp 146-147°C; UV λmax 263, 293, and 328 nm; IR (KBr) cm−1: 3341, 2957, 2761, 1611, 1597, 1506, and 1463; and HRESIMS m/z 418.1270 calc. For C21H22O9, 418.1264, and 1H and 13C NMR data see  6 was isolated as dark yellow amorphous powder. The positive HREIMS analysis showed an ion peak at m/z 436.1374 which corresponded to the molecular formula C21H24O10. The IR showed absorption of hydroxyl (3341 cm−1) and aromatic rings (1647 and 1489 cm−1) functions. Aliphatic proton was assigned to ABX system at δH 4.70 , δH 2.99 , and δH 2.81  suggesting the presence of -CO-CH (OH)-CH2– moiety. Compound 6 showed the presence of a glucopyranosyl moiety which was linked to C-3′ according to the HSQC correlation of anomeric proton at δH 5.02  with C-3′ (δC 164.03). The anomeric configuration was assigned as β for the glucopyranosyl group from their coupling constants. The downfield shift of C-4′ at δC 144.59 was indicative that it has a hydroxy group. Aromatic protons H-3′ (δH 6.68), H-5′ (δH 6.44), and H-6′ (δH 7.71) suggested a 2′,4′-dioxygenation in a trisubstituted ring, which is corroborated by the NOESY and the HMBC spectrum  of A ring indicates 1,4-disubstitution, whereas the chemical shift of C-4 (δC 164.03) reveals hydroxyl group at this position. Therefore, the structure of compound 6 was identified as α,4,4′-trihydroxydihydrochalcone-2′-O-β-d-glucopyranoside (Compoundspectrum . The AA′ranoside .7) [8) [9) [10) [11) [12) [ sandwicensis (13) [In addition, seven known compounds were also isolated  by compa7) , (3R)-5,7) [8) , flemich [8) [9) , neohesp[9) [10) , hespere10) [11) , aspalat11) [12) , and sansis (13) . 1–5 isolated from E. polystachya are given in  6 displayed lower scavenging value and the data are not presented here. Chalcones 4, 3, 5, and 1 showed significant scavenging activity in DPPH radical with IC50 values of 10.21 to 19.39 μg/mL. These results suggested that these compounds showed the highest hydrogen-donating capacity towards the DPPH radical.Radical scavenging capacities of compounds 4 (33.17 μg/mL) was found to exhibit the highest hydroxyl radical scavenging effect followed by 5 (36.52 μg/mL) and 3 (37.45 μg/mL). Scavenging of hydrogen peroxide by the isolated compound was found to be better compared to the ascorbic acid (IC50 443.76 μg/mL). Among the tested flavonoids, the best scavenging of hydrogen peroxide activity was presented by 3 (IC50 80.73 μg/mL), 4 (IC50 87.68 μg/mL), and 1 (IC50 91.13 μg/mL). The presence in the –OH group in ring A and -OCH3 in ring B showed good antioxidant activity on scavenging of hydrogen peroxide activity compared to electron-withdrawing substituents at paraposition.Hydroxyl radicals are found in living systems that react with almost all biomolecules as they are high oxidant. The orders of reactivity of the flavonoids were similar to that of DPPH. Compound 4, 3, and 5.Superoxide radical is a free radical harmful to cellular components, precursor for many reactive oxygen species. Superoxide radical scavenging capacity followed an activity order of chalcones50 of 25.47 to 75.04 μg/mL. Compound 4 showed the highest scavenging effects on nitric oxide, followed by 3, 5, and 1. Reactive nitrogen species  are formed during the reaction with superoxides or oxygen which are responsible for altering the functional and structural cellular components.As evident from 2+ complex decreased in the order of 4 > 5 > 3 > 1 > 2.The ability to chelate and deactivate transition metals is an important mechanism of antioxidant activity, which catalyzes Fenton-type reactions and hydroperoxide decomposition. Therefore, it was considered of importance to study chelating ability of the isolated compound with the metal. The chelating activity of flavonoids to suppress the formation of ferrozine-Fe 4 and 3 and pterocarpan 5 also showed good significant scavenging activities of Trolox (16.23 to 20.34 nM) indicating their good antioxidant effect.Chalcones2O2 was also determined. Exposure of H2O2 to BSA produced a decrease of free thiol groups level. Results are shown in 2O2 in a dose-dependent manner. Among the five compounds tested, 4 showed the strongest antioxidant activity at a concentration of 300 mg/mL preventing the 97% of the depletion of protein thiol groups. These results indicated that hydrophilic antioxidant moiety in the compounds has the ability to scavenge H2O2 reducing thiol groups oxidation in BSA.The inhibition of the isolated compound on the oxidation of BSA induced by H 4, 3, and 1 indicated that chalcones having electron donor group in the phenyl ring A in orthoposition to carbonyl moiety showed better antioxidant activity. Moreover, the activity decreasing obviously with glycosidation such as only 1 showed a moderate inhibitory activity, and the other glycoside 2 showed a low inhibitory effect. However, pterocarpan showed a good antioxidant activity in various oxidation systems.The antioxidant activity in all tested assays indicates that the compound with chalcone skeleton showed the best inhibitory activity when the dihydrochalcone as in compoundsAntioxidants compounds have been shown to delay, inhibit, and prevent the oxidation, possibly through the mechanisms interacting with biological systems as scavenging free radicals, absorbing oxygen radicals, and chelating of the metal ions . 1–5 to decrease the oxidative stress in cells was determined. It is well known that concentrations of palmitate at 500 μm increase oxidative stress and stimulate ROS production in H9c2 cell. H2O2 model has been used to determine the oxidative stress because it can generate superoxide anion radical. Pretreatment with flavonoids for 1 hr at 20 μM significantly decreased ROS production preventing oxidative stress in H9c2 cell and palmitate-induced ROS production using DCFH-HA (for H2O2−) and DHE (for O2−) test. The oxidative stress reduction potential of 1–5 is given in μg/mL flavonoids to reduce the oxidative stress was compared with that of 25 μg/mL of ascorbic acid. The values of the mean fluorescent intensity indicated that the isolated compound significantly reduces the increase of ROS in palmitate-induced cells. The antioxidant effectiveness in vitro of the isolated compound can be possibly due to their ability to act as free radical scavengers, quenchers of singlet O2 formation, to complex with prooxidant metal ions and reducing agents [The ability ofg agents . 1–5. There was a significant increase of lipid peroxide in the liver, kidney, and pancreas in diabetes mice. However, the administration of 1–5 improved these levels in the treated groups with respect to the diabetic control group as shown in  1–5, and increases in radical scavenging enzymes of CSH-Px, SOD, GSH, and CAT have also been observed in liver, pancreas, and kidney in the group of diabetic animals treated.There were no changes of blood glucose level after 30 days of administration of 1–5 reduced levels of these enzymes leading to hepatoprotective effect.Elevation of serum biomarker enzymes such as total protein, SGPT, SGOT, ALP, and TB was observed in diabetic mice indicating deterioration in liver function  which map < 0.05) diminution in lipid peroxidation levels, in liver and kidney compared with diabetic control, and the observed reduction of TBARS towards normal levels , α,3,2′,4′-tetrahydroxy-4-methoxy-dihydrochalcone-3′-C-β-glucopyranosy-6′-O-β-d-glucopyranoside (2), 7-hydroxy-5,8′-dimethoxy-6′α-l-rhamnopyranosyl-8-(3-phenyl-trans-acryloyl)-1-benzopyran-2-one (3), 6′7-dihydroxy-5,8-dimethoxy-8(3-phenyl-trans-acryloyl)-1-benzopyran-2-one (4), and 9-hydroxy-3,8-dimethoxy-4-prenylpterocarpan (5). The isolated compounds were evaluated for their antioxidant potentials in in vitro and in vivo assay finding that compound 4 is the most potent antioxidant. Our data indicates that isolated 1–5 from the bark of Eysenhardtia polystachya have an ability to reduce oxidative stress under diabetic conditions, prevent and/or delay the onset renal, pancreatic, and hepatic damage through decreasing of lipid peroxidation, antioxidant properties, and increasing radical scavenging enzymes activity, also reduce intracellular reactive oxygen species, and they consequently could alleviate complications of diabetes. In addition, the antioxidant properties of compounds 1–5 are a promising strategy for ameliorating therapeutic effects by avoiding disorders in the normal redox reactions in healthy cells.In this study were isolated and characterized five biologically active compounds from bark of


T, was isolated from cold waters of Llullaillaco Volcano Lake (6170 m asl) in Chile. Phylogenetic analysis based on 16S rRNA gene sequences identified strain DB165T as belonging to the genus Subtercola in the family Microbacteriaceae, sharing 97.4% of sequence similarity with Subtercola frigoramans DSM 13057T, 96.7% with Subtercola lobariae DSM 103962T, and 96.1% with Subtercola boreus DSM 13056T. The cells were observed to be Gram-positive, form rods with irregular morphology, and to grow best at 10–15 °C, pH 7 and in the absence of NaCl. The cross-linkage between the amino acids in its peptidoglycan is type B2γ; 2,4-diaminobutyric acid is the diagnostic diamino acid; the major respiratory quinones are MK-9 and MK-10; and the polar lipids consist of phosphatidylglycerol, diphosphatidylglycerol, 5 glycolipids, 2 phospholipids and 5 additional polar lipids. The fatty acid profile of DB165T (5% >) contains iso-C14:0, iso-C16:0, anteiso-C15:0, anteiso-C17:0, and the dimethylacetal iso-C16:0 DMA. The genomic DNA G+C content of strain DB165T was determined to be 65 mol%. Based on the phylogenetic, phenotypic, and chemotaxonomic analyses presented in this study, strain DB165T (= DSM 105013T = JCM 32044T) represents a new species in the genus Subtercola, for which the name Subtercola vilae sp. nov. is proposed.A novel actinobacterium, strain DB165 Microbacteriaceae are widely distributed in terrestrial and aquatic environments or associated with macroorganisms  in Chile, one of the highest-elevation lakes on Earth. According to its distinct properties, strain DB165T is proposed as the type strain of the new species Subtercola vilae.In this study, we characterise strain DB165T was obtained from a water sample collected at the Llullaillaco volcano lake  on 18 January 2013, using R2A medium (DIFCO) supplemented with 18 g agar l−1. Pure cultures were obtained after three successive transfers of single colonies to R2A medium plates. Stock cultures were maintained in SGG medium containing 10 g starch, 10 g glucose, 10 ml glycerol (99.7% v/v), 5 g soy peptone, 2.5 g corn steep solids, 2 g yeast extract, 3 g CaCO3, 1 g NaCl, and 18 g agar in 1 l deionised water  for long term storage at − 80 °C.Strain DB165−1.Gram-staining was prepared using the Color Gram 2 kit , following the manufacturer’s protocol. Endospore staining was performed using the green malaquite method and light microscopy (Schaeffer and Fulton S. boreus DSM 13057T and S. frigoramans DSM 13057T were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) and cultured under the same conditions as strain DB165T for comparative purposes.The reference strains T, S. boreus DSM 13057T and S. frigoramans DSM 13057T were examined using API ZYM, API 20E and API 50CH , following the manufacturer’s recommendations. The effect of sodium chloride  and pH  on the growth was tested according to Kutzner .DNA was extracted using the DNeasy Blood & Tissue kit (QIAGEN). The G+C content was calculated from the genome sequence, which was determined with Nextseq 500 (Illumina). The quality of the sequences was checked and filtrated using Trimmomatic   with modifications. The 16S rRNA gene sequence was amplified by PCR using PureTaq Ready-To-Go PCR beads  and sequencing according to Gärtner et al. .T was aligned with sequences of 22 selected type strains of the family Microbacteriaceae, including members of the genera Subtercola, Frondihabitans and Agreia, and in addition Cellulomonas carbonis KCTC 19824T as outgroup using SINA , but are pale yellow after growth at 5 °C for 2–3 weeks. Optimum growth is observed at 10–15 °C (range from 5 to 28 °C). No growth occurs at 30 °C. Strain DB165T tolerates only low concentrations (up to 0.9%) of NaCl and grows best in the absence of NaCl. The pH range for growth is from 5 to 8, with an optimum at pH 7. Cells show no motility and form no spores, they are irregular short rods of 0.5 µm width and 1.0–1.2 µm length, Gram-positive and have an irregular shape as seen under SEM. Some of the cells are thicker at the ends. Occasionally, coccoid cells were observed . The molar ratio of alanine:glycine:glutamic acid:DAB was 1.1:1.0:0.04:1.7. Instead of glutamic acid, high amounts of 3-hydroxyl-glutamic acid were found. Assuming that much of the glutamic acid is replaced by 3-hydroxyl-glutamic acid, and despite this replacement, the amino acid composition is consistent with peptidoglycan type B2γ. The major isoprenoid quinones of strain DB165T were identified as MK-9 (47%) and MK-10 (39%). Minor amounts of MK-11 (6%) and MK-8 (4%) were also found to be present. The G+C content of the genomic DNA of the strain DB165T was determined to be 65.0 mol%.The polar lipids of the strain DB165T were identified as iso- and anteiso-saturated C15 and C16 fatty acids with anteiso-C15:0 (50%), iso-C16:0 (17%) and iso-C16:0 DMA (17%) as major components  matches to different Subtercola species in a range of 96.1–97.4% of similarity, i.e., S. frigoramans DSM 13057T (97.4% similarity), S. lobariae DSM 103962T (96.7% similarity) and S. boreus DSM 13056T (96.1% similarity). However, it also showed high similarity to Frondihabitans species, including Frondihabitans peucedani DSM 22180T (96.8% similarity) and Frondihabitans australicus DSM 17894T (96.6% similarity). The phylogenetic analysis .The genome of strain DB165Subtercola species are characterised by a peptidoglycan type B2γ with DAB as diamino acid, MK-9 and MK-10 as major respiratory quinones, and a similar polar lipids profile   and MK-11 (21%) as major menaquinones, but in addition 13% of MK-9 .d-sorbitol, d-sucrose, d-melibiose, glycerol, l-arabinose, d-xylose, methyl-β-d-xylopyranoside, d-galactose, d-glucose, d-fructose, d-mannose, l-rhamnose, d-mannitol, amygdalin, arbutin, esculin, salicin, d-cellobiose, d-maltose, d-sucrose, d-trehalose, d-melezitose, gentiobiose and d-turanose. The cell-wall peptidoglycan is type B2γ with DAB as the diagnostic amino acid and 3-hydroxyl-glutamic acid instead of glutamic acid. Major menaquinones are MK-9 and MK-10. Polar lipids comprise phosphatidylglycerol, diphosphatidylglycerol, 5 unidentified glycolipids, 2 unidentified phospholipids and 5 unidentified lipids. The major cellular fatty acids are anteiso-C15:0, iso-C16:0, anteiso-C17:0, and iso-C14:0, while C14:0 and C16:0 are found only in traces. Major dimethylacetals are iso-C16:0 DMA, anteiso-C15:0 DMA and anteiso-C17:0 DMA, while C16:0 DMA and iso-C15:0 DMA are present in trace amounts. The G + C content of the DNA of the type strain is 65.0 mol%.Cells are short, irregular rods 0.5 µm wide and 1.0–1.2 µm long. Colonies are golden yellow, circular convex. Growth occurs chemoheterotrophically under oxic conditions. Optimum growth is at 10–15 °C (range from 5 to 28 °C), at pH 7 (range from pH 5 to 8) and in the absence of NaCl. Cells produce esterase C4, esterase lipase C8, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α- and β-glucosidase, tryptophan deaminase, tryptophanase and acetoin. Weak activity is observed for valine arylamidase, cysteine arylamidase, trypsin, and α- and β-galactosidase. Carbon sources used under oxic conditions include inositol, T (= DSM 105013T = JCM 32044T) was isolated from Llullaillaco volcano lake in Chile. The GenBank/EMBL/DBBJ accession number for the 16S rRNA of strain DB165T is MF276890.The type strain DB165


By contrast, α-Syn caused little or no effect on S1P2 receptor-mediated signalling. Both wild-type and α-Syn(A53T), a mutant found in familiar PD, caused uncoupling of S1P1 receptor, although α-Syn(A53T) showed stronger potency in uncoupling. Moreover, S1P1 receptor-mediated β-arrestin signal was unaltered by α-Syn(A53T). These results suggest that exogenous α-Syn modulates S1P1 receptor-mediated signalling from both Gi and β-arrestin signals into β-arrestin-biased signal. These findings uncovered a novel function of exogenous α-Syn in the cells.Parkinson’s disease (PD) is the second most common neurodegenerative disorder. The presence of α-synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, is the cytopathological hallmark of PD. Increasing bodies of evidence suggest that cell-to-cell transmission of α-Syn plays a role in the progression of PD. Although extracellular α-Syn is known to cause abnormal cell motility, the precise mechanism remains elusive. Here we show that impairment of platelet-derived growth factor-induced cell motility caused by extracellular α-Syn is mainly attributed to selective inhibition of sphingosine 1-phosphate (S1P) signalling. Treatment of human neuroblastoma cells with recombinant α-Syn caused S1P type 1 (S1P Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder after Alzheimer’s disease. The pathological hallmarks of PD are selective loss of the dopaminergic neurons in the substantia nigra pars compacta and the presence of α-synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, which develop in the cell body of affected neurons in both idiopathic127i.e., α-Syn pathology is initiated in the peripheral nervous system and olfactory bulb, ascends toward the brainstem and into the midbrain such as substantia nigra, and then eventually spreads to the forebrain as suggested by “Braak’s hypothesis”α-Syn with 140 amino acids is highly expressed in neurons and enriched in synaptic terminals suggesting a role in synaptic function and plasticity91113161–5) and triggers diverse cellular processes, including cell angiogenesis, cardiac development, immunity, cell motility, neurotransmitter release and endosome maturation232425Our laboratory has recently demonstrated that extracellular α-Syn causes inhibition of platelet-derived growth factor (PDGF)-induced chemotaxis in human neuroblastoma-derived SH-SY5Y cells through selective suppression of rac1 activation which is required for cell migration211 receptor–selective uncoupling from inhibitory G-protein (Gi) while other signalling events including S1P2 receptor-mediated signalling remain intact. A possible interpretation of α-Syn-induced changes in cellular signalling in conjunction with pathophysiological relevance is discussed herein.From this background we have studied the extracellular effects of α-Syn on S1P signalling-mediated cell motility using both functional and fluorescence resonance energy transfer (FRET)-based structural analyses. Here we show that extracellular α-Syn causes S1P1 receptor-specific blocker, W146, inhibited PDGF-induced chemotaxis to an extent similar to α-Syn(A53T) treatment . SphK1-siRNA caused inhibition of PDGF-induced chemotaxis by 30% as compared with the control siRNA  and yellow fluorescent protein (YFP) were separately fused in the same S1P1 receptor molecule , a mutant α-Syn found in familiar PD, caused more potent effects than a wild-type α-Syn with the same concentration (1 μM). On the other hand, when the cells expressing S1P2 receptor-CFP and Gγ-YFP were stimulated with S1P, the agonist-induced G-protein dissociation occurred. However, wild-type α-Syn and α-Syn(A53T) treatment showed no significant effects on S1P2 receptor-mediated G-protein subunit dissociation   causes S1Pficiency , extrace1 receptor antagonist W146-sensitive manner  abrogated the S1P1 receptor/Giα-caused inhibition of forskolin-stimulated adenylate cyclase, supporting the premise that α-Syn(A53T) caused S1P1 receptor to uncouple from Gi protein. α-Syn(A53T) itself had no inhibitory effect on forskolin-stimulated adenylate cyclase activity. In contrast, S1P-induced increase in cellular Ca2+ through S1P2 receptor, which was inhibited by JTE-013  on endogenous Gi function was assessed next. When cells were treated with S1P, forskolin-stimulated adenylate cyclase activity was potently inhibited  in a S1Pe manner , evaluat JTE-013 , was ins JTE-013 . These rn system  and 3.T on endo1 receptor was assessed using an S1P1 receptor FRET tool. S1P-induced conformational changes in the receptor were not influenced by α-Syn(A53T) treatment -induced respiratory burst. We therefore undertook a study to compare α-Syn(A53T) with PTX for the ability to cause uncoupling of fMLP receptor. As reported previously1 receptor was measured. S1P caused association of β-arrestin with S1P1 receptor in a time-dependent manner with the maximum level in 30 min -treated cells showed a decrease in a cell surface S1P1 receptor after S1P stimulation to an extent similar to that in the control cells (1 receptor using an antibody to detect extracellular epitope of the receptor. Stimulation of cells with S1P caused a decrease in endogenous cell surface S1P1 receptor both in control and α-Syn(A53T)-treated cells to a similar extent  causes uncoupling between S1P1 receptor and Gi protein, leaving β-arrestin signal unchanged.Since G-protein-coupled receptors are known to utilise several other downstream signalling molecules such as β-arrestin as well as G-proteinn 30 min . Importalisation . It shouRecent studies have revealed that α-Syn can be released from cultured cells by exocytosis161 receptor-selective uncoupling from Gi as determined by both functional  although in the proapoptotic conditions such as a higher dose (10 μM) of α-Syn or prolonged time (42 hr) treatment the mRNA level of SphK1 decreased, while that of SphK2 increased (data not shown).In the present studies we have shown that extracellular α-Syn causes S1Pnctional  and FRETnctional . It has 1 receptor because fMLP-induced respiratory burst was insensitive to α-Syn(A53T) in differentiated HL-60 cells, although it was completely inhibited by PTX, which acts on Giα subunit directly  association (data not shown). This suggests that α-Syn(A53T) may exert its action indirectly or with the aid of other molecules. In this context it has been shown that the translocation of S1P1 receptor to caveolin-enriched microdomains is necessary for the subsequent efficient signalling1 receptor and Akt is important in endothelial barrier enhancement in human pulmonary endothelial cells. It has been suggested that α-Syn has an ability to interact with gangliosides in the cholesterol and sphingolipid-rich membrane microdomains known as lipid rafts, and has a potency to alter the functions of several signalling molecules at the raft regions1 receptor function.As for the site of action of α-Syn(A53T), it may not act on Gi protein but on S1Pdirectly . α-Syn (1 receptor was unaffected by α-Syn(A53T)   and its yn(A53T) . It has  studies . These ci.e., their enzymatic products are ceramide. Ceramide is shown to be involved in exosomal vesicle formation in multivesicular endosomes (MVEs)1 receptor by S1P, a further metabolite of ceramide, on MVEs has an essential role in the cargo sorting into exosomes1 receptor from Gi caused by extracellular α-Syn may inhibit exosomal release of α-Syn, which results in the accumulation of α-Syn inside the cells, a pathological hallmark of PD. Further studies on the mechanism underlying extracellular α-Syn-caused uncoupling of S1P1 receptor from Gi will be the keys to understand the pathogenesis of sporadic PD.Although the discovery of hereditary forms of PD has contributed greatly to understand the pathogenesis of the disease, that of sporadic PD, the majority forms of PD, is still elusive. It has recently been revealed that mutations in the GBA and SMPD1 genes are risk factors for PD. GBA encodes the lysosomal enzyme glucocerebrosidase that catalyses the breakdown of the glycolipid glucosylceramide to ceramide and glucose. GBA mutations, when homozygous, lead to Gaucher’s disease, while predispose to PD when heterozygousS1P was purchased from Enzo Life Sciences; PTX, forskolin and dibutyryl cAMP from Wako Pure Chemical Industries; fMLP and cytochrome c were from Sigma Aldrich. W146 and JTE-013 were from Cayman Chemical Company. Other reagents and chemicals were of analytical grade.1 receptor-CFP, Giα-CFP, Gβ and Gγ-YFP plasmid constructs were prepared as described previously2 (mS1P2) receptor (GenBank accession number NM_010333.4) cDNA was amplified from mouse brain cDNA, which had been reverse transcribed from fetal mouse brain mRNA (Invitrogen) by PCR  to make a C-terminally CFP-fused construct in pECFP-N1. A one-molecular FRET probe for detection of cAMP, Epac1-camps, was constructed as reported previously1 receptor, EYFP was inserted in the third intracellular loop of murine S1P1 receptor between Lys243 and Ala244. Briefly, the cDNA encoding Met1 to Lys243, Ala244 to Ser382 of mS1P1, or EYFP was amplified by PCR using 5′-CGGGAATTCGCCACCATGGTGTCCACTAGCATCC-3′ and 5′-ATAGGATCCCTTGGAGATGTTCTTGCG-3′, 5′-GCTCTAGAGCCAGTCGCAGTTCTGAG-3′ and 5′-CGGAATTCGGGAAGAAGAATTGACGTTTCCAG-3′, or 5′-ATAGGATCCATGGTGAGCAAGGGCGAG-3′ and 5′-GCTCTAGACTTGTACAGCTCGTCCATG-3′, respectively. Each cDNA fragment was treated with EcoRI and BamHI, XbaI and EcoRI or BamHI and XbaI, respectively, and these three fragments were inserted into EcoRI-cut pECFP-N1. All the constructs were verified by sequencing.Human α-Syn was amplified and subcloned into the bacterial expression vector pET3a. For α-Syn(A53T), alanine 53 was mutated to a threonine using a QuikChange site-directed mutagenesis protocol. mS1P1 receptor;sense 5′-GGGCAAGGCCUUGCAGCUCdTdT-3′ and antisense 5′-GAGCUGCAAGGCCUUGCCCdTdT-3′ for human SphK1;sense 5′-UUCUCCGAACGUGUCACGUdTdT-3′ and antisense 5′-ACGUGACACGUUCGGAGAAdTdT-3′ for control.For RNA interference following oligonucleotides  were used: Sense 5′-GGAGAACAGCAUUAAACUGdTdT-3′ and antisense 5′-CAGUUUAAUGCUGUUCUCCdTdT-3′ for human S1PSH-SY5Y cells were transfected with the siRNAs using Lipofectamine RNAiMAX according to the manufacturer’s instructions .Recombinant α-Syn and α-Syn(A53T) were expressed in E. Coli and purified as described previously2. HL-60 cells were grown in RPMI 1640 medium  containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% CO2. Cells were plated onto 35 mm glass-bottom culture dishes (MatTek) before transfection. Transient transfection was carried out using FuGENE HD (Promega). All experiments were performed 2 to 3 days after transfection.SH-SY5Y cells  were maintained in DMEM/F-12 medium  containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% CO1 receptor-YFP were incubated at 37 °C with or without 1 μM α-Syn(A53T) for 18 hr in serum-free DMEM/F-12 and treated with or without 100 nM S1P for 1 hr. Cells were then treated with sulfo-NHS-SS-biotin (Thermofisher Scientific) at 4 °C for 30 min and solubilised with lysis buffer , followed by centrifugation at 10,000 × g for 15 min at 37 °C. Biotinylated S1P1 receptor-YFP was pulled-down by streptavidin agarose beads  for 1 hr at 4 °C and the amount of biotinylated cell-surface S1P1 receptor-YFP was directly measured using an EnSpire plate reader (Perkin-Elmeer). Values represent means ± s.e.m. of 3 independent experiments carried out in triplicate. To detect endogenous S1P1 receptor internalisation, SH-SY5Y cells (1 × 106 cells) were serum-starved in the absence or presence of 1 μM α-Syn(A53T) for 18 hr. After cell stimulation with S1P, cells were chilled on ice and harvested by using Cell Dissociation Buffer . Cells were washed with PBS containing 1% sodium azide and 1% BSA and incubated with 1 μg/ml of anti-S1P1 receptor antibody conjugated with Alexa 488 , which detects endogenous cell surface S1P1 receptor from outside of the cells, at 4 °C for 1 hr, followed by washing and analysed by flow cytometry .SH-SY5Y cells expressing S1P6 cells) using NucleoSpin RNA II (Macherey-Nagel) according to the manufacturer’s instructions. 1 μg RNA was used for reverse transcription . Quantitative PCR was performed with SYBR Premix (Takara) on ABI Prism 7000. Expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control for normalisation. The primer sequences (sense and antisense) were as follows: for human S1P1 receptor, 5′-TTCCTGGTGTTAGCTGTGCTCAAC-3′ and 5′-TCGCTTGAATTTGCCAGCAGAGTC-3′; for human S1P2 receptor, 5′-TGCGCCATTGTGGTGGAAAACC-3′ and 5′-TTGCCCAGAAACAGGTACATTGCC-3′; for human S1P3 receptor, 5′-AGCAGCAACAATAGCAGCCACTC-3′ and 5′-AGTGCTGCGTTCTTGTCCATGATG-3′; for human S1P4 receptor, 5′-AACTGCCTGTGCGCCTTTGAC-3′ and 5′-ATCACCAGGCAGAAGAGGATGTAGC-3′; for human S1P5 receptor, 5′-TTCCTGCTGCTGTTGCTCGAC-3′ and 5′-TTCAGAAGTGAGTTGGCCATGGC-3′; for human GAPDH, 5′-GCCATCAATGACCCCTTCATT-3′ and 5′-TCTCGCTCCTGGAAGATGG-3′.Total RNA was extracted from SH-SY5Y cells  for 18 hr and treated with various reagents. Cells were then fixed and each area of interest was subjected to FRET analysis with acceptor photobleaching method using a LSM 510 META with a 63 x oil plan-apochromat objective. Following excitation at 458 or 514 nm, CFP emission with a 475–525-nm band-pass barrier filter or YFP emission with 530–600-nm band-pass barrier filter, respectively, was collected. An area of interest was selected for photobleaching of YFP. An automated acquisition protocol was then used, which recorded pre- and post-bleaching images using 458 nm excitation at 8% laser power to limit photobleaching, with a bleaching of the selected area with 100%, 514 nm laser power with 50 iterations (acceptor photobleaching). FRET was resolved as an increase in the CFP (donor) signal after photobleaching of YFP (acceptor). FRET efficiency (E) can be determined from the relative fluorescence intensity of the energy donor (CFP) before (Ipre) and after (Ipost) photobleaching of the energy acceptor (YFP): E = 1 − (Ipre/Ipost).SH-SY5Y cells were transiently cotransfected with S1P2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 10 mM glucose and 0.2% BSA with or without 1 μM α-Syn(A53T). Cell suspension (106 cells/tube) was treated with 0.1 μM fMLP and 50 μM cytochrome c at 37 °C for 10 min followed by rapid centrifugation. The cell superoxide anion production was estimated by measuring the reduction of cytochrome c as the increase in absorbance at 550 nm using a spectrophotometer.HL-60 cells were cultured for 48 hr with 0.2 mM dibutyryl cAMP to induce differentiation followed by serum starvation in the absence or presence of 1 μM α-Syn(A53T) or 100 ng/ml PTX for 18 hr. Differentiated HL-60 cells were suspended in the HEPES-buffered medium consisting of 10 mM HEPES/NaOH (pH 7.4), 130 mM NaCl, 4.7 mM KCl, 1 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 10 mM glucose with or without 1 μM α-Syn(A53T) at 37 °C for 20 min and washed. Cells were pretreated with 10 μM W146 or 10 μM JTE-013 or without (control) for 10 min. In some experiments cells were treated with 1 μM α-Syn(A53T) for 10 min. The glass bottom dish was mounted on IX70 fluorescence microscope (Olympus) and Fluo-4 fluorescence was recorded. Excitation wavelength was set to 490 nm and emission was recorded at 535 nm. After taking basal level fluorescence for 30 s, 100 nM S1P was added and the change of fluorescence was monitored at a frequency of 1 Hz. The acquired Fluo-4 emission signal for each cell was normalised to the basal signal and plotted against time.SH-SY5Y cells seeded on glass bottom dish were incubated with or without 1 μM α-Syn(A53T) for 18 hr in serum-free DMEM/F-12. Cells were loaded with 2 μM Fluo-4 AM (Dojindo Laboratories) in HEPES-buffered medium consisting of 10 mM HEPES/NaOH (pH 7.4), 130 mM NaCl, 4.7 mM KCl, 1 mM CaCl1 receptor-FLAG and β-arrestin 2-YFP were serum-starved for 18 hr with or without 1 μM α-Syn(A53T) and stimulated with 100 nM S1P for the indicated time periods. Cells were then treated with 2 mM Di(N-succinimidyl) 3,3′-Dithiodipropionate (DSP)  for 20 min at room temperature, followed by solubilisation with lysis buffer  and protease inhibitor cocktail ). Cell lysates were cleared by centrifugation at 13,000 × g for 15 min and immunoprecipitated by anti-FLAG beads . The immunoprecipitates were separated by SDS-PAGE and co-immunoprecipitated β-arrestin 2-YFP was detected by rabbit monoclonal anti-β-arrestin 2 antibody  in immunoblotting.SH-SY5Y cells expressing S1PSH-SY5Y cells were serum-starved in the absence or presence of 1 μM wild-type α-Syn or α-Syn (A53T) for the indicated time periods. Cells were stained with 4,6-diamidino-2-phenylindole-2-HCl (DAPI) and chromatin condensation during apoptosis was visualised by using LSM510 confocal microscope.Results are expressed as means ± s.e.m. Data were analysed by t-test. P-values < 0.05 were considered significant.How to cite this article: Zhang, L. et al. Extracellular α-synuclein induces sphingosine 1-phosphate receptor subtype 1 uncoupled from inhibitory G-protein leaving β-arrestin signal intact. Sci. Rep.7, 44248; doi: 10.1038/srep44248 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


A and B). In the crystal, molecules of each conformer make a face-to-face type dimeric molecular aggregate with two pairs of C—H⋯π hydrogen bonds (A) or a pair of (sp2)C—H⋯O hydrogen bonds (B). The dimeric molecular aggregates composed of same conformers are linked to each other into a chain through π–π stacking interactions (A) or a pair of C—H⋯π hydrogen bonds (B) along the b axis. The chains are aligned along the c axis by weak van der Waals interactions or (sp2)C—H⋯O=O hydrogen bonds, and they are alternately stacked along the a axis.The asymmetric unit of the title compound contains two independent molecules . The anthracene ring system is connected to the 2,7-dimethoxynaphthalene core in a twisted manner, with dihedral angles of 86.38 (5) and 79.36 (8)°, respectively, for conformers A and B. In the crystal, face-to-face type dimeric molecular aggregates of each conformer are observed. The dimer of conformer A is formed by two pairs of C—H⋯π interactions, and that of conformer B by a pair of (sp2)C—H⋯O hydrogen bonds. The dimers of conformer A are linked to each other via a π–π stacking interaction between the anthracene rings to form a chain along the b axis and the chains are aligned along the c axis, forming a sheet structure. The dimers of conformer B are connected to each other via a couple of C—H⋯π interactions to form a chain along the b axis. The chains are aligned along the c axis through (sp2)C—H⋯O=C hydrogen bonds, forming a sheet parallel to the bc plane. The sheets of conformers A and B are alternately stacked along the a axis via two kinds of intermolecular (sp2)C—H⋯O=C hydrogen bonds.The asymmetric unit of the title compound, C Furthermore, the two aroyl groups at peri-positions of the naphthalene ring core are situated in positions very close to each other. Accordingly, a coplanar alignment of the aromatic rings in a molecule is not possible in peri-aroylnaphthalene compounds because of their highly congested molecular arrangement. On the other hand, the spatial organization around the ketonic carbonyl groups of a diaryl ketone structure is supposed to be rather loose compared to that of directly combined aromatic ring systems, as shown in the rotation barrier for an analogous compound in solution ° between the anthracene and naphthalene ring systems, which is slightly larger than that of 79.36 (8)° for conformer B.There are two independent molecules in the asymmetric unit of the title compound. The conformers, labeled A, a pair of C—H⋯π (anthracene) interactions and another pair of (methoxy)C—H⋯π  ones are observed  Å between the C12–C13/C18–C20/C25 and C13–C18 rings]. The chains of conformer A are aligned along the c axis by weak van der Waals interactions, forming a sheet structure parallel to the bc plane. In the dimer of conformer B, a pair of (anthracene)C—H⋯O(methoxy) hydrogen bonds are observed C—H⋯O=C hydrogen bonds C—H⋯O=C and (anthracene)C—H⋯O=C hydrogen bonds , AlCl3  was gradually added. After stirring for 10 min, 2,7-dimethoxynaphthalene  was added to the CH2Cl2 solution. The reaction mixture was stirred in ice-bath for 6 h, then poured into ice-cold water. The aqueous layer was extracted with chloroform (30 ml × 3) and the combined extracts were washed with 2 M aqueous NaOH solution (30 ml × 3) followed by washing with brine (20 ml × 3). The organic layer thus obtained was dried over anhydrous MgSO4. The solvent was removed under reduced pressure to give a cake. The title compound was separated from the crude product by column chromatography (eluent: toluene); isolated yield 42%. Yellow needle single crystals suitable for X-ray diffraction were obtained by repeated crystallization from ethyl acetate solution.To a solution of 9-anthroyl chloride  and CH1H NMR  δ: 3.00 , 3.86 , 6.90 , 7.14 , 7.42 , 7.45 , 7.76 , 7.87 , 8.0–8.02 , 8.12 , 8.49  p.p.m. 13C NMR  δ: 55.281, 56.178, 102.94, 111.11, 117.38, 124.07, 124.88, 125.08, 125.46, 126.15, 128.44, 128.51, 130.14, 131,11, 133.42, 134.32, 138.67, 138.76, 159.36, 160.43, 201.25 p.p.m. IR (KBr): 1640(C=O), 1618(Ar), 1511(Ar), 1250(OMe) cm−1. HRMS (m/z): [M + H]+ calculated for C27H21O3, 393.1491, found, 393.1494. m.p. = 444.8–446.9 K.Uiso(H) = 1.2 Ueq(C). Rigid bond restraints (DELU) were applied to O3—C8, C1—C2, C2—C3, C4—C5, C5—C6, C7—C8 and C9—C10 in the naphthalene moiety.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016018077/is5460sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016018077/is5460Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016018077/is5460sup3.psORTEP of molecule A for Fig 1. DOI: Click here for additional data file.10.1107/S2056989016018077/is5460sup4.psORTEP of molecule B for Fig 1. DOI: Click here for additional data file.10.1107/S2056989016018077/is5460Isup5.cmlSupporting information file. DOI: 1516088CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The aim of this research was to synthesize an 18F–labeled silicon containing exendin-4 peptide (18F-2) was prepared via one-step nucleophilic substitution of a silane precursor with 18F–fluoride in the presence of acetic acid and K222. 18F-2 was then administered to tumor-bearing mice for PET imaging and ex vivo biodistribution experiments.18F-2 was produced in a radiochemical yield (decay corrected) of 1.5% and a molar activity of max. 16 GBq/μmol. The GLP-1R positive tumors were clearly visualized by PET imaging. Biodistribution studies showed reduced uptake of 18F-2 in the kidneys compared to radiometal labeled exendin-4 derivatives. The radiotracer showed specific tumour uptake which remained steady over 2 h.18F-2, is a potential probe for imaging GLP-1R positive tumors.This exendin-4 analogue,  Semi-preparative radio-HPLC purification was performed with a reversed-phase column . Both analytical and semi-preparative HPLC chromatograms were obtained by use of an Agilent 1100 system equipped with multi-UV-wavelength (measuremt were done at 254 nm) and Raytest Gabi Star detectors and Gina Star software.His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Nle-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Pro-Ala-Pro-Pro-Pro-Ser-Lys-(N6–2-(4-(di-tert-butylsilyl)phenyl)acetyl)-NH2(precursor1). Precursor 1 was provided by Peptide Specialty Laboratories GmbH, Heidelberg, Germany as lyophilized white solid. The product was re-analyzed by the MS-Service at LOC of ETH Zurich. HRMS (ESI-MALDI) calcd. For [C207H321N52O62Si]+: 4555.3328, found: 4555.3496. The purity (> 95%) of precursor 1 was confirmed by analytical HPLC .His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Nle-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Pro-Ala-Pro-Pro-Pro-Ser-Lys-(N6–2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl)-NH2). Precursor 1  was dissolved in DMSO (200 μL). This solution was added to a mixture of KF , Kryptofix 222 , and K2CO3 . Glacial acetic acid  was added and the resulting suspension was heated at 70 °C for 30 min. An aliquot of the reaction mixture was purified by analytical HPLC  to afford 2 (RT = 11.18 min). The product was analyzed by LC-MS: m/z calcd.: 4573.3, found: 4573.0 ([M + H]+).18F]fluoride was produced on an IBA Cyclone 18/9 cyclotron by irradiation of 98% enriched [18O]H2O (2.0 mL) using an 18-MeV proton beam via the [18O18F] nuclear reaction. [18F]Fluoride was trapped on a preconditioned anion-exchange resin cartridge , water (10 mL) and air (10 mL)). The cartridge was eluted with a solution of K222 (5.0 mg) and K2CO3 (1.0 mg) in acetonitrile (1.2 mL) and water (0.3 mL). The fluoride was dried by azeotropic distillation of acetonitrile at 100 °C under vacuum with a stream of nitrogen. The azeotropic drying process was repeated three times with acetonitrile (1 mL).No-carrier-added [1  and glacial acetic acid (10 μL) in anhydrous DMSO (150 μL) was added to the dry K[18F]F/K222 complex  and heated at 110 °C for 15 min. After cooling at room temperature for 5 min, a mixture of acetonitrile/H2O + 0.1% TFA  was added to the reaction vial and the diluted mixture was purified by semi-preparative radio-HPLC using 0.1% TFA/water solution (solvent A) and 0.1% TFA/acetonitrile (solvent B) as the solvent system at a flow rate of 4 mL/min and with a gradient as follows: 0–15 min 95% A, 15–40 min 55% A. The fraction containing 18F-2 was collected into a solution of 1 mM glutamic acid and 0.5% TFA/water solution (30 mL) and immobilized on a C18 cartridge , Machery-Nagel). After washing with 0.9% NaCl-water solution (20 mL), 18F-2  was eluted with a solution of 1 mM HCl/ethanol  into a vial containing 0.15 M PBS solution (0.5 mL). The mixture was neutralized by adding a 1 mM NaOH aqueous solution (100 μL) and the ethanol was evaporated at 95 °C with a gentle stream of nitrogen. The identity of 18F-2 was confirmed by comparison with the HPLC retention time of the nonradioactive reference compounds (RT = 11.19 min) using analytical radio-HPLC . For in vivo applications, 18F-2 was passed through a sterile filter into a sterile, pyrogen-free vial. Hydrolytic stability of 18F-2 was tested after the addition of PBS to an aliquot of the ethanolic solution of the product. Typical concentration of tracer was 30 MBq in 100–120 μL. The concentration of ethanol was equal or less than 5% providing a maximal ratio of EtOH:PBS of 1:19. This mixture was analyzed by HPLC at different time points.The radiosynthesis was performed manually in a hot cell using a manipulator. A solution of precursor 1 and native exendin-4 were determined using a displacement assay on CHL cells stably transfected with the GLP-1 receptor gene  at 90–95% confluence. The test compound solutions and [125I]-exendin-4 (9–39)  were added to all well plates. The final concentrations of the test compound in the wells were in the range of 1 pM to 1 μM. For the total binding no cold peptide was added. The total volume was adjusted with medium containing 0.1% BSA to 1 mL. The cells were incubated at 4 °C for 1 h and subsequently washed twice with cold phosphate buffered saline (PBS) and solubilised with 0.5 mL 1 M NaOH (2×). The radioactivity was measured in a γ-counter . The 50% inhibitory concentration (IC50) values were calculated using GraphPad Prism  fitting the data with nonlinear regression using least squares fit. Experiments were performed on triplicate samples and repeated three times.The binding affinities to the GLP-1 receptor for both the precursor compound 7.4) was carried out by the shake-flask method in analogy to a published procedure  and 1–octanol (0.5 mL) at room temperature. The mixture was equilibrated for 15 min in an overhead shaker and further centrifuged . Aliquots (50 μL) of both phases were analyzed in a γ–counter . The partition coefficient was expressed as the ratio between the radioactivity concentrations (cpm/mL) of the 1–octanol and the PBS phase. Values represent the mean ± standard deviation of five determinations from one experiment.The determination of distribution coefficient ) under 2%–3% isoflurane anaesthesia. Ex vivo biodistribution experiments were conducted three weeks after the inoculation. PET imaging experiments were performed five weeks after the inoculation.Animal studies complied with Swiss laws on animal protection and husbandry and were approved by the Veterinary office of the Canton Zurich. After an acclimatization period, tumor xenografts were produced in 6-week old female CD1 nu/nu mice by subcutaneous injection in both shoulder regions of CHL-GLP-1 receptor positive cells (8 × 10n = 12) were injected intravenously with 18F-2 . The animals were sacrificed at 30 min, 1 h and 2 h post injection (n = 4 for each time point). To determine specific binding an additional group of tumor-bearing mice (n = 4) received nonradioactive precursor 1  co-injected with 18F-2  and were sacrificed 1 h post injection. Organs and tissues of interest were collected and weighed, and the amount of radioactivity was determined in a γ-counter  to calculate percentage uptake (% injected dose per gram of tissue). Statistical significance was calculated using Student t-test . P values of less than 0.05 were considered statistically significant.Tumor-bearing mice  via lateral tail vein. Anaesthesia was induced with 5% isoflurane (Abbott) in O2/air 5 min before PET/CT acquisition. Depth of anaesthesia and temperature were controlled as described by Honer et al.  were calculated as a ratio of tissue radioactivity concentration (kBq/cm3) and injected activity dose per gram body weight (kBq/g) at the scan start. Percentage injected dose per gram of tissue (%ID/g) was calculated using SUV values: SUV / body weight [g] × 100% = %ID/g.Two tumor bearing mice were injected with 18F–Labeling of silicon containing exendin-4 analogue proceeded as depicted in Fig. 18F]F/K222 complex and was finished within 60–75 min and maximal 18F–incorporation yield was 6%. After HPLC purification and formulation 180–270 MBq of 18F-2 were obtained. The radiochemical yield was 1.0–1.5% (decay corrected) and [18F]2 was afforded in ≥ 95% radiochemical purity and molar activity of 12–16 GBq/μmol (end of synthesis). The hydrolytic stability test of 18F-2 in PBS showed that no defluorination occurred within 2 h (data not shown). The log D7.4 value of 18F-2 as a measure of hydrophilicity was determined by the shake-flask method and amounted to −1.38 ± 0.06 (n = 5), indicating good water solubility.50 value determined for precursor 1 was 169 ± 19 nM, which is approximately fifteen-fold higher compared to exendin-4 (10.4 nM ± 1.6 nM).The ICex vivo biodistribution data of 18F-2 in xenograft-bearing mice. Thirty minutes after injection, tumor uptake was 15 ± 7%ID/g. It essentially remained steady over the measured period of time and was reduced significantly by a blocking dose of precursor 1 (P < 0.03). Tracer uptake in the lungs was constant and in the same range as in the tumors. Radioactivity uptake in the pancreas and the stomach was lower and decreased slightly over time. The GLP-1 receptor-positive organs lung and stomach revealed a significantly reduced tracer uptake under blocking conditions. Tracer accumulation in blood was high (9.7 ± 1.1%ID/g) at 30 min p.i. and decreased after 120 min p.i. (3.3 ± 0.8%ID/g), resulting in increased tumor to blood ratio (4 ± 2 at 120 min p.i.), but indicating a relatively slow blood clearance of the tracer. Bone uptake remained steady at ~ 2%ID/g and highest accumulation of the radiotracer was found in kidneys (49 ± 18%ID/g at 60 min p.i.).Table 18F-2 is shown in Fig. 18F-2 was in the kidneys (SUVkidneys: 4.1 and 5.0) followed by the tumors (SUVtumors: 2.2 and 2.3) and liver (SUVliver: 1.4 and 1.6). Tumor to background ratio was ~ 3.A PET/CT image of a tumor bearing mouse after injection of 99mTc and 111In being the most widely used radionuclides . Therefore, the development of 18F–exendin tracer would have a high impact in imaging β-cell derived diseases.It was shown by different groups that radiolabeled exendin derivatives are good tracers for targeting the GLP-1R  was achieved by using 4 mg (878 nmol) of the silane precursor. The labeling reaction mixture contained unidentified side-products, which were easily removed by HPLC purification, however, for 18F-2 and precursor 1 no clear-cut baseline separation was achieved. The consequence of this was that some amount of the silane precursor 1 was still found in the formulated product solution, which partly contributed to the rather low molar activity of 12–16 GBq/μmol.The silicon-based building block can be used for a direct one-step 18F-2 is a hydrophilic peptide with a logD7.4 value of −1.38 ± 0.06, however compared to other radiolabeled exendin derivatives . A fifteen-fold lower affinity value was obtained with 1 compared to parent exendin-4.ex vivo biodistribution, the highest tumor uptake of 15%ID/g was observed at 30 min p.i. Beyond this time point tumor retention was still high and amounted to 13%ID/g at 2 h p.i. The uptake in the kidneys was in the range between 30 to 50%ID/g which was dramatically lower compared to the kidney uptake of radiometal labeled compounds, which is normally much higher than 100%ID/g  during the PET studies and during the ex vivo biodistribution studies (1.3 pmol). Since more unlabeled peptide was injected during the PET studies, the GLP-1 receptors in the lungs might have been saturated. The tumor uptake could significantly be decreased by co-injection of unlabeled exendin-4, suggesting specific binding of the radioligand to GLP-1 receptors.18F–Al-NOTA, where the kidney uptake stays high (Xu et al. 18F]2 may potentially find application in imaging insulinoma.An important aspect is the kidney uptake which is very high with the radiometal labeled exendin derivatives (Jodal et al. 18F with the silicon based method retaining the main biological properties like affinity and exhibit a favourable biodistrution with a specific uptake in the receptor positive pancreas and tumor. The kidney uptake is markantly reduced compared to radiometal labelled Exendin-4 derivatives.We could show that the Exendin-4 can be labelled with


II atom is each of the title compounds is coordinated by two dithiocarbamate ligands and a pyridyl-N atom. The resultant NS4 donor set approximates a square-pyramid and trigonal-bipyramid, for the solvated and unsolvated structures, respectively. In the solvate, amide-N—H⋯O(dimethylformamide) hydrogen-bonds define a three-molecule aggregate while in the unsolvated structure, amide⋯amide hydrogen-bonding leads to a supramolecular chain.The Zn 2(C3H6NS2)4(C14H14N4O2)]·2C3H7NO (I) and [Zn2(C7H14NS2)4(C14H14N4O2)] (II), each feature a bidentate, bridging bipyridyl-type ligand encompassing a di-amide group. In (I), the binuclear compound is disposed about a centre of inversion, leading to an open conformation, while in (II), the complete molecule is completed by the application of a twofold axis of symmetry so that the bridging ligand has a U-shape. In each of (I) and (II), the dithiocarbamate ligands are chelating with varying degrees of symmetry, so the zinc atom is within an NS4 set approximating a square-pyramid for (I) and a trigonal-bipyramid for (II). The solvent dimethylformaide (DMF) molecules in (I) connect to the bridging ligand via amide-N—H⋯O(DMF) and various amide-, DMF-C—H⋯O interactions. The resultant three-molecule aggregates assemble into a three-dimensional architecture via C—H⋯π interactions. In (II), undulating tapes sustained by amide-N—H⋯O(amide) hydrogen bonding lead to linear supramolecular chains with alternating molecules lying to either side of the tape; no further directional interactions are noted in the crystal.The title structures, [Zn In this connection, isomeric di-amide structures of the general formula (n-NC5H4)CH2N(H)C(=O)—C(=O)N(H)CH2(C5H4N-n), for n = 2, 3 and 4, hereafter abbreviated as nLH2, have long attracted interest for their potential to form supramolecular tapes. For example, as realized in the two-dimensional structure formed in the 1:1 co-crystal of 4LH2 and the conformer, bi-functional 1,4-di-iodobuta-1,3-diyne 2]BF4}n by a combination of Ag←N bonds for the tetrahedral silver(I) atom, provided by bidentate bridging ligands, where the latter are also connected via concatenated {⋯HNC2O}2 synthons 2Br]·Br·H2O}n  dithiocarbamates functionalized with hydrogen-bonding potential, i.e. Zn[S2CN(R)CH2CH2OH]2, for R = alkyl, CH2CH2OH. As discussed in more detail in the Database survey, none of these crystals exhibited self-association of the amide residues. For example, in the crystal of binuclear {Zn[S2CN(Me)CH2CH2OH]2}2(3LH2), supramolecular chains are constructed as a result of hydroxy-O—H⋯O(hydroxy) hydrogen bonding that leads to the formation of sterically unencumbered 28-membered {⋯HOC2NCSZnSCNC2O}2 synthons. Two chains inter-weave through these rings and are held in place by hydroxy-O—H⋯O(amide) hydrogen bonding  dithiocarbamates with the view of ‘turning-off’ putative hydroxy-O—H⋯O(amide) hydrogen bonding. Herein, the crystal and molecular structures of [Zn(S2CNMe2)]2(3LH2)·2DMF  and {Zn[S2CN(n-Pr)2]2}2(3LH2), (II)via amide⋯amide hydrogen bonding in (I)A similar coordination/hydrogen-bonding arrangement is found in the three-dimensional assembly in crystals of {2(pyridine)·pyridine for R = Me and Et CH2CH2OH]2(3-hydroxypyridine)  hydrogen bonds in Zn[S2CN(i-Pr)CH2CH2OH]2 CH2CH2OH]2(hmta) 2]2}2 with pyrazine CH2CH2OH]2}2(3LSH2) is recrystallized from acetonitrile  hydrogen bonding leading to a two-dimensional array. When DMF is diffused into an acetonitrile solution of the same compound, one hydroxy group hydrogen bonds to the DMF-O while the other hydroxyl group self-associates to form a supramolecular chain. In the present study, when additional hydrogen-bonding functionality is not present, the amide groups are able to self-assemble as shown in Fig. 3i.e. variable coordination geometries, flexible conformations of the bridging ligands and different hydrogen-bonding potential, more systematic studies in this area are warranted.The investigation of zinc(II) dithiocarbamates, Zn(S2CNMe2)2 and 3LH2 in DMF; m.p. 479–481 K. Crystals of (II)2CN(n-Pr)2]2 and 3LH2 in a MeOH/EtOH solution.Crystals of (I)Uiso(H) set to 1.2–1.5Ueq(C). The N-bound H atoms were located in difference-Fourier maps but were refined with a distance restraint of N—H = 0.88±0.01 Å, and with Uiso(H) set to 1.2Ueq(N). Owing to poor agreement, two reflections, i.e.  I, II, global. DOI: 10.1107/S2056989017012956/hb7704Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017012956/hb7704IIsup3.hklStructure factors: contains datablock(s) II. DOI: 1573823, 1573822CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The unique capabilities of gamma-delta (γδ) T cells to recognize cells under stressed conditions, particularly infected or transformed cells, and killing them or regulating the immune response against them, paved the way to the development of promising therapeutic strategies for cancer and infectious diseases. From a mechanistic standpoint, numerous studies have unveiled a remarkable flexibility of γδ T cells in employing their T cell receptor and/or NK cell receptors for target cell recognition, even if the relevant ligands often remain uncertain. Here, we review the accumulated knowledge on the diverse mechanisms of target cell recognition by γδ T cells, focusing on human γδ T cells, to provide an integrated perspective of their therapeutic potential in cancer and infectious diseases. More than three decades after the discovery of gamma-delta (γδ) T cells , the res+ and Vδ3+ T cells that are highly reactive to cytomegalovirus (CMV) infection  γδ T cell subtype in humans, seemingly expand in the periphery in response to microbial or stress-induced phosphorylated antigens  was first proposed, with biophysical and structural data in support . Howevervia γδ TCRs. This notwithstanding, it is clear that γδ T cells also rely on “NK-like” mechanisms for tumor cell recognition, using receptors such as 2B4 and NKG2D, originally thought to be specific to NK cells.Besides sensing PAgs, γδ T cells seemingly recognize transformed cells through proteins that are expressed at the cell surface in a stress-induced manner. Some examples are typically endogenous proteins, such heat shock protein 60 –17 or FI+ γδ T cells were able to develop non-MHC-restricted cytotoxicity against lymphoma cells  A/B  production  A/B –33, 62 oMIC) A/B , 50, 63 or cells , 59. Ouror cells . Notwithor cells , 38. In or cells , 31. A soduction , 22.+, Vδ2+, and Vδ1−Vδ2− γδ T cell subsets  was also shown to be widely expressed in Vδ1 subsets ; and mas subsets , 65. DNA subsets , acute  γδ T cells (+ T cells activated with TCR agonists and IL-15 (or IL-2); and both receptors enhanced γδ T cytotoxicity against tumor target cells  that recognize a distinct set of tumor-associated ligands, such as B7-H6 or BAT3 . Thus, o T cells . A very  T cells ; and sho T cells . In our et cells , 73. Amoandidate , a very andidate , known fandidate . AdditioMycobacterium tuberculosis -4-hydroxy-3-methyl-but-2-enyl pyrophosphate, an intermediate of the 2-C-methyl-karyotes , 92, 93.rculosis –81 or P.lciparum . Besideslciparum ; OXYS anlciparum , 21; andlciparum , 21.+ T cells has been clearly shown to bind CD1d loaded with the self-lipid sulfatide , thus allowing it to recognize endothelial cells infected with CMV  expression; and established the proof-of-concept in leukemia xenograft models . All clinical trials have thus far concentrated on Vγ9Vδ2 T cells, probably due to their relative abundance in the peripheral blood and especially the availability of FDA-approved drugs, such as zoledronate and pamidronate, that allow their activation and expansion in vivo . Vγ9Vδ2 in vivo  and has in vivo ] that doin vivo –108. Thiin vivo , 110. As T cells . Howevert models . We furtAS, BL, and BS-S conceived and wrote the manuscript. AS and BL contributed equally to the manuscript.BS-S is a co-founder and shareholder of Lymphact—Lymphocyte Activation Technologies S.A. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The preparation and the crystal structure of octane-1,8-diaminium 4,4′-azinodibenzoate monohydrate are reported. 8H22N22+·C14H8N2O42−·H2O, represents a pseudo-polymer ionic material, resulting from the self-organizing behavior of 4,4′-azinodibenzoate dianions and doubly protonated, 1,8-diaminium-octane cations in aqueous solution. The asymmetric unit consists of two halves of octane 1,8-diaminium cations , a 4,4′-azinodibenzoate anion [dihedral angle between the aromatic rings = 10.22 (4)°] and a water molecule of crystallization. One of the cations is in a fully extended linear conformation while the second one has a terminal C—C—C—N gauche conformation. In the crystal, the cations, anions and water molecules are linked into a three-dimensional network via a complex pattern of charge-assisted N—H⋯O and O—H⋯O hydrogen bonds.The title salt, C The resulting orange oblong crystals in the form of thin narrow leaves up to 1 cm long were gently removed from the liquid phase and air-dried on filter paper.Crystals of the title compound were obtained by the dropwise addition with intensive stirring of 5 ml of 0.10 Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018006187/hb7747sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018006187/hb7747Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018006187/hb7747Isup3.cmlSupporting information file. DOI: 1839024CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


This transformed the MgO tunneling barrier from a body-centered cubic (b.c.c) crystallized layer into a mixture of b.c.c, face-centered cubic, and amorphous layers and rapidly decreased the tunneling-magnetoresistance (TMR) ratio. The p-MTJ spin valve with a nanoscale-thick top Co2Fe6B2 free layer could prevent the Pt atoms diffusing into the MgO tunneling barrier during ex situ annealing at 400 °C because of non-necessity of a Pt buffer layer, demonstrating the TMR ratio of ~143 %.For the perpendicular-magnetic-tunneling-junction (p-MTJ) spin valve with a nanoscale-thick bottom CoThe online version of this article (doi:10.1186/s11671-016-1637-9) contains supplementary material, which is available to authorized users. Δ), and switching current density (Jc) 4-SyAF layer/Co/Ru spacer/Co/Pt/upper [Co/Pt]6-SyAF layer/Ta/Ru top electrode. On the other hand, as shown in Fig. 2Fe6B2 free layer were deposited with vertically stacked layers from the SiO2 substrate, W/TiN bottom electrode/Ta buffer layer/Pt (3.0 nm) seed layer/upper [Co/Pt]6-SyAF layer/Co/Ru spacer/Co/Pt/lower [Co/Pt]4-SyAF layer/Co/b.c.c bridge layer/Co2Fe6B2 (1.0 nm) pinned layer/MgO (1.15 nm) tunnel barrier/Fe (0.4 nm)/Co2Fe6B2 (1.0 nm) free layer/b.c.c capping layer/Ta/Ru top electrode. The b.c.c metal has a lattice constant of 3.16 Å [2Fe6B2 layer to obtain a higher TMR ratio [CoFeB/MgO-based p-MTJ spin valves with a [Co/Pt]layer/Pt .0 nm see2Fe6B2 free layer and ~158 % for the p-MTJ spin valve with a nanoscale-thick top Co2Fe6B2 free layer, indicating the TMR ratio did not depend on the p-MTJ spin valve structure. However, the TMR ratio for the p-MTJ spin valve with a nanoscale-thick bottom Co2Fe6B2 free layer rapidly decreased from ~160 to ~72 % when the ex situ annealing temperature increased from 350 to 400 °C. Otherwise, the TMR ratio for the p-MTJ spin valve with a nanoscale-thick top Co2Fe6B2 free layer slightly decreased from ~158 to ~143 % when the ex situ annealing temperature increased from 350 to 400 °C. These results imply that the TMR ratio for the p-MTJ spin valve with a nanoscale-thick bottom Co2Fe6B2 free layer (~88 %) decreased by approximately six more than that for the p-MTJ spin valve with a nanoscale-thick top Co2Fe6B2 free layer (~15 %).The TMR ratio for p-MTJ spin valves depending on the p-MTJ spin valve structure was estimated by using current-in-plane tunneling (CIPT) at room temperature , as showM-H) curves measured by a vibrating sampling magnetometer (VSM) at room temperature, as shown in Fig. 2Fe6B2 free layers consisted of four magnetic layers as shown in Fig. 2Fe6B2 free layer (a), Co2Fe6B2 pinned layer (b), lower SyAF layer (c), and upper SyAF layer (d). In Fig. 2Fe6B2 pinned layer (b) was ferro-coupled with the lower SyAF layer (c) while the lower SyAF layer (c) was antiferro-coupled with the upper SyAF layer (d) n-SyAF layer.To find out why the i-PMA magnetic moment of the Coaterials . The dif2Fe6B2 free and pinned layers and the b.c.c crystallinity of the MgO tunneling barrier should be prevented as much as possible from degrading. The design of the p-MTJ spin valve structure is a key to meet these goals. For the p-MTJ spin valve with a nanoscale-thick bottom Co2Fe6B2 free layer  of both Coyer Fig. , when thyer Fig.  since it