In this paper, removal of Cr (VI) and Ni (II) ions from aqueous solution was investigated using an amino-functionalized magnetic Nano-adsorbent (Fe3O4-NH2) was synthesized by compositing Fe3O4 with 1, 6-hexanediamine for removal of Cr (VI) and Ni (II) ions from aqueous solution. The adsorbent was characterized by Scanning Electron Microscope (SEM), Transmission Electron Microscopy (TEM), powder X-Ray Diffraction (XRD), and Vibrating Sample Magnetometry (VSM). Also, the effects of various operational parameters were studied.An amino-functionalized magnetic Nano-adsorbent  and Ni (II) ions revealed that the process followed the Langmuir isotherm and the maximum adsorption capacity was 232.51 mg g−1 for Cr (VI) at pH = 3 and 222.12 mg g−1 and for Ni(II) at pH = 6 at 298 °K. Besides, the kinetic data indicated that the results fitted with the pseudo-second-order model (R2: 0.9871 and 0.9947 % for Cr (VI) and Ni (II), respectively. The results of thermodynamic study indicated that: standard free energy changes (ΔGɵ), standard enthalpy change (ΔHɵ), and standard entropy change (ΔSɵ) were respectively −3.28, 137.1, and 26.91 kJ mol−1 for Cr (VI) and −6.8433, 116.7, and 31.02 kJ mol−1 for Ni (II). The adsorption/desorption cycles of Fe3O4-NH2 indicated that it could be used for five times.According to our finding, Fe3O4-NH2 could be removed more than 96 % for both Cr (VI) and Ni (II) ions from aqueous solution and actual wastewater.The selected metals’ sorption was achieved mainly via electrostatic attraction and coordination interactions. In fact, Fe Amonectively . Due to ectively –11. Mostectively . Until nectively , amino-fectively , and Lewectively . Howeverectively –18. In tectively .3O4 particles, with strong magnetic properties, low toxicity, and easy separation, it could be used as a sorbent for removing heavy metals [As described above, due to the high specific surface area created through grafting of appropriate organic amino-groups on inorganic magnetic Fey metals , 20. Anoy metals , 21.3O4-NH2) developed by grafting amino-groups onto the surfaces of Fe3O4 nanoparticles and used nanocompostie as the adsorbent for removal of Cr (VI) and Ni (II) from aqueous solution. The adsorbent was characterized by Transmission Electron Microscopy (TEM), powder X-Ray Diffraction (XRD), Vibrating Sample Magnetometry (VSM), Scanning Electron Microscope (SEM), and zeta-potential measurement. The effects of pH, initial concentrations of Cr (VI) and Ni (II), adsorption kinetics, thermodynamics, and adsorption isotherm were studied, as well.In this study, we prepared a novel amino-functionalized magnetic Nano-adsorbent (Fe3 · 6H2O), potassium dichromate, ethanol, 1,6-hexanediamine, ethylene glycol, nickel (II) chloride hexahydrat (NiCl2.6H2O), sodium hydroxide, hydrogen chloride, which were of analytical grade, were purchased from Merck, Germany and were used without further purification. Potassium dichromate (99 %) and nickel (II) chloride hexahydrat (99 %) were used for preparation of Cr (VI) and Ni (II) solution. Additionally, doubly distilled deionized water was used throughout the work.Anhydrous sodium acetate, iron (III) chloridehexahydrat (FeCl3O4-NH2) was prepared according to hydrothermal reduction method. In doing so, a solution of 1, 6-hexanediamine (13 g), anhydrous sodium acetate (4.0 g), and FeCl3 · 6H2O as a single Fe ion source (2.0 g) was added to ethylene glycol (80 mL). The above mixture was stirred at 50 °C under vigorous stirring for 30 min. Then, this solution was heated at 198 °C in a Teflonlined autoclave for 6 h. Thereafter, the mixture was cooled down to room temperature. The magnetite nanoparticles were collected with a magnet and were then washed with water and ethanol (3 times) to effectively remove the solvent and unbound 1, 6-hexanediamine. Finally, the amino-functionalized magnetic Nano-adsorbent (Fe3O4-NH2) was dried in a vacuum oven at 50 °C before characterization and application [3O4-NH2 were showed by SEM . Besides, the magnetic property (M–H loop) of the typical magnetic nanoparticles bound with 1, 6-hexadiamine at 25 °C was characterized by VSM . The crystal structure and phase purity of Fe3O4-NH2 were also examined by XRD  using Cu Kα radiation (λ = 0.1541 nm) at 2θ, 30 kV, and 30 mA. Finally, the TEM image of Fe3O4-NH2 was examined using TEM, Model EM10C-100KV .Amino-functionalized magnetic Nano-adsorbent (Felication . The siz−1 concentrations and 0.05 g of Fe3O4-NH2. The mixtures were stirred (200 rpm) at room temperature from 10 to 90 min. After adsorption, Fe3O4-NH2 with adsorbed Cr (VI) and Ni (II) was separated from the solution under the external magnetic field. The concentrations of Cr (VI) and Ni (II) ions in the solutions were measured by an Inductive Coupled Plasma ).The absorption experiments were conducted in 1000 ml Erlenmeyer flasks containing 50 ml Ni (II) and Cr (VI) solutions at 5 to 100 mg L3O4-NH2 doses (0.1 to 0.3 g/L), initial concentrations of Cr (VI) and Ni (II) (5 to 100 mg/L), and temperatures (298.15 to 338.15 °K). Besides, each experiment was carried out in duplicate. The removal of Cr (VI) and Ni (II) by Fe3O4-NH2 and removal efficiency have been figured by equations in Table In order to determine the effects of various factors, the experiments were performed at different Fe3O4-NH2 were recorded. The SEM of Fe3O4-NH2 image has been shown in Fig. 3O4-NH2 was much smaller than that of naked particles, confirming the coating of 1, 6 hexanediamine [The SEM, VSM, XRD, and TEM of Feediamine .Fig. 1SE3O4 and Fe3O4-NH2 were essentially super-paramagnetic. Fe3O4-NH2 and Fe3O4 have a magnetization saturation value of 73.25 and 91.57 emu g−1, respectively. According to Fig. 3O4-NH2 was dispersed in water. In addition, it could be collected by external magnetic field and be re-dispersed through slight shaking, making the solid and liquid phases separate easily.The magnetic hysteresis loops measured at room temperature has been illustrated in Fig. 3O4-NH2 have been shown in Fig. 3O4-NH2 were examined by XRD using a Cu Kd radiation (λ = 0.1541 nm) at 2θ of 30.1°, 35.5°, 43.1°, 53.4°, 57.0°, and 62.6° corresponding to their indices; i.e., 220, 311, 400, 422, 511, and 440, at 30 kV and 30 mA. The particle size was obtained via XRD analysis through Debye-Sherrer’s formula [The XRD patterns of Fe formula : D = K λ3O4-NH2 was under 90 nm. Additionally, the sharp and strong peaks of the products revealed its appropriate crystallinity. Moreover, the six characteristic peaks of Fe3O4 showed that amino-groups did not cause any measureable alter in the phase property of Fe3O4 cores. Therefore, the amino-groups were fixed on the surface of Fe3O4 cores, making a core-shell structure. In other words, binding and amino-functionalization (NH2) occurred only on the surface of Fe3O4 cores to form a core–shell structure [Where λ is the wavelength of the X-rays, θ is the diffraction angle, and β is the corrected full width. The result of size distribution demonstrated that the size of the prepared Fetructure .3O4-NH2 has been shown in Fig. 3O4-NH2 particles were multi-dispersed with an average diameter of around 25 nm. It has been reported that magnetic particles of less than 30 nm would show paramagnetism [The TEM image of Feagnetism .Fig. 5TE3O4-NH2 by varying C0 of Cr (VI) and Ni (II) ions at 5, 25, 50, and 100 mg L−1. The results have been presented in Figs. 3O4-NH2 concentration of 5 mg L−1 decreased from 98.02 to 36.85 % for Cr (VI) and increased from 46.21 to 93.03 % for Ni (II) with increasing the pH from 2.0 to 9.0. This can be justified by the fact that for a fixed adsorbent dosage, the total available adsorption sites would be relatively settled. Thus, increasing the initial Cr (VI) and Ni (II) concentrations led to a decrease in the adsorption percentage of the adsorbate [The effect of initial concentration on the adsorption properties was intensively studied for Fe2O3 [3O4-NH2. The plot of pH initial vs. pH final depicted that the pHzpc was 5.8 for Fe3O4–NH2. Hence, at pH >5.8, the surface charge of Fe3O4-NH2 was negative and the electrostatic interactions between the metal ions and the adsorbent enhanced. Considering Ni (II), the interaction between the adsorbents and the Ni (II) ions might be defined by Equations To assess the effect of pH, the study was conducted from pH 2 to 9 for both Cr (VI) and Ni (II) ions. The maximum sorption was perceived at pH = 6 for Ni (II), but at pH = 3 for Cr (VI). The adsorption of Cr (VI) at lower pH levels was also observed in other magnetic materials, such as the mesoporous magnetic ɤ-Fe2O3 . pH valu3O4-NH2 amino-groups in the solution have been presented in Equation 2 to be protonated was weakened at higher pH levels, resulting in more –NH2 on the surface of the adsorbent to coordinate with Ni (II). At higher pH levels, OH− in the solution is competitively adsorbed by amino-groups (−NH2), and the electrostatic adsorption is prevailed gradually compared to coordination. Considering Cr (VI), a large number of H+ exists under acidic conditions (pH levels: 2–3.5), causing amino-groups (−NH2) to be protonated to NH3+ more easily and electrostatic attraction to occur between these two oppositely charged ions (Equation (The protonation/deprotonation reactions of the FeEquation ) [14, 273O4-NH2 were gained at pH = 3 for Cr (VI) and pH = 6 for Ni (II) with the initial concentrations of 5 to 100 mg L−1. The relevant equations for kinetic, equilibrium, and thermodynamic studies have been shown in Table 2), and separation factor (RL) for the adsorption of Cr (VI) and Ni (II) on Fe3O4-NH2 have been summarized in Table L). The plot of Ce vs. (Ce/qe) for Fe3O4-NH2 gave a straight line with correlation coefficients of 0.998 and 0.994 for Cr (VI) and Ni (II), respectively. The qmax and KL were derived from the slope and intercept of the line, respectively. The adsorption capacities  using the Langmuir isotherm equation were as follows: qm Ni (II) (232.15) > qm Cr (VI) (222.12). Considering the larger adsorption capacity of Fe3O4-NH2 attributed to the amino-groups modified on the surface of Fe3O4-NH2, the amino-groups played a very important role in the adsorption process of Cr (VI) and Ni (II) in aqueous solution. This implies that increasing the percentage of nitrogen in the Fe3O4-NH2 could increase the value of qm. The calculated RL values for adsorption of Cr (VI) and Ni (II) were also 0.03–0.39 and 0.02–0.34, respectively, which fall between 0 and 1. Thus, the adsorption of Cr (VI) and Ni (II) onto NH2- Fe3O4 was favorable. However, the correlation coefficients (R2 > 0.99) and RL (0 < RL < 1) proved that the Langmuir isotherm fitted better for adsorption of Cr (VI) and Ni (II) on NH2- Fe3O4. On the other hand, the value of 1/n shows whether the adsorption is suitable for the Freundlich isotherm [3O4-NH2 for Cr (VI) and Ni (II) was high (qm for Ni (II) = 232.51 mg/g−1 at pH = 6 and qm for Cr (VI) = 222.12 mg/g−1 at pH = 3) compared to other adsorbents. Afkhami et al. also reported that the adsorption capacity of DNPH-γ-Al2O3 for Ni (II) was 18.18 (mg g−1) at pH = 5 and that the process followed the Langmuir isotherm [3O4-GS revealed that the process followed the Langmuir isotherm and that the maximum adsorption capacity was 158.5 mg g−1 at pH = 6 [1 and K2 constants were determined experimentally from the slope and intercept of straight-line plots. The value of qe.c earned from the pseudo-second-order model was 28.25 mg g−1 for Cr (VI) and 25.97 mg g−1 for Ni (II) ions, which perfectly corresponded to the experimental values of qe (24.25 and 25.12 mg g−1) for Cr (VI) and Ni (II) ions. Overall, the pseudo-second-order model (R2: 0.9871 and 0.9947 % for Cr (VI) and Ni (II), respectively) was more efficient compared to the pseudo-first-order model (R2: 0.8422 and 0.8862 % for Cr (VI) and Ni (II), respectively). Because all the correlation coefficients were higher than 0.98 %, Cr (VI) and Ni (II) adsorption onto Fe3O4-NH2 might take place through a chemical process involving valence forces through sharing or exchange of electrons [3O4adsorbent, R2 value of Ni (II) was 0.998 at the optimum pH of 5.5. Therefore, the results showed that Ni (II) adsorption on Fe3O4 could be followed by the Freundlich model [3O4-NH2,the adsorption studies were accomplished at 298.15 to 313.15 °K. The negative values of ΔGθ at different temperatures, positive value of ΔSθ, and positive value of ΔHθ during the adsorption of Cr (VI) and Ni (II) on Fe3O4-NH2 indicated that the adsorption was spontaneous, increased randomness at the solid-solution interface, and was endothermic in nature [θ and ΔSθ values [θ) and standard entropy change (ΔSθ), which were related to distribution coefficient (KD), were calculated and presented in Table θ and ΔSθ values, standard free energy changes (ΔGθ) for Fe3O4-NH2 were estimated. The results indicated that adsorption of Cr (VI) and Ni (II) on Fe3O4-NH2 could be followed spontaneously, was endothermic, and was entropy favored in nature. The positive value of ΔSθ proved increase in the randomness at the solid-solution interface during the adsorption of Cr (VI) and Ni (II) on Fe3O4-NH2. This indicated that the amino-functionalized magnetic Nano-adsorbent (Fe3O4-NH2) could be regarded as an efficient and low cost adsorbent. The results of thermodynamic study in our research indicated that ΔGɵ, ΔHɵ, and ΔSɵ were respectively −3.28, 137.1, and 26.91 kJ mol−1 for Cr (VI) and −6.8433, 116.7, and 31.02 kJ mol−1 for Ni (II). Shen et al. conducted a similar study using adsorbent DETA-NMPs and disclosed that ΔGɵ, ΔHɵ, and ΔSɵ were −13.7, 8.41, and 72.83 kJ mol−1, respectively for Cr (VI) [ɵ, ΔHɵ, and ΔSɵ were −1.599, 8.438, and 83.1, respectively for Ni (II) adsorption on Nano-HAP [Adsorption isotherms of Feisotherm . The valisotherm . In anott pH = 6 . The parch model . In ordeNano-HAP .Table 2T3O4-NH2 make it a potential applicant for Cr (VI) and Ni (II) removal. Considering the larger adsorption capacity of Fe3O4-NH2 attributed to the amino-groups modified on the surface of Fe3O4-NH2, the amino-groups played a very important role in the adsorption process of Cr (VI) and Ni (II) in aqueous solution. This indicated that the increase of nitrogen percentage in Fe3O4-NH2 could result in an increase in the value of qm. Similar results were also obtained by Shen et al. [Overall, simple preparation, fast separation, and high adsorption capacity of Fen et al.  and Zhaon et al. .3O4-NH2 is of particular importance. Since the adsorption of Cr (VI) and Ni (II) onto Fe3O4-NH2 highly depends on the solution pH, desorption of the two heavy metals can be achieved by adjusting the pH. In the present study, the adsorption reversibility of Cr (VI)-laden Fe3O4-NH2 and Ni (II)-laden Fe3O4-NH2 was examined using NaOH  and HNO3 . For desorption studies, the metal-adsorbed modified Fe3O4 nanoparticles were first washed by ultrapure water for three times to remove the unadsorbed metals loosely appended to the adsorbent. When the concentration of NaOH and HNO3 was increased, the removal efficiency of desorption increased, as well. The best result was achieved with 2 min sonication in the presence of 0.2 mol L−1 NaOH and 0.05 mol L−1 HNO3. Finally, the Fe3O4-NH2 was dried in an oven (at 50 °C) during regeneration. In our study, each sorption/desorption process experienced a base and a heat treatment. The adsorption/desorption cycle results showed that the Fe3O4-NH2 could be reused for 5 times. Besides, the results presented in Fig. 3O4-NH2 maintained more than 76.19 % of its original Cr (VI) adsorption capacity and 77.13 % of its original Ni (II) adsorption capacity. Therefore, the great reusability Fe3O4-NH2 demonstrated its good potential for practical application.For practical application of a cost-effective adsorbent for Cr (VI) and Ni (II) removal, desorption of metal ions from adsorbent and regeneration of Fe−1 Cr (VI) and Ni (II) were spiked with tap water and industrial wastewater for evaluating the practical application of Fe3O4-NH2. The initial concentration, pH, and removal efficiencies of Cr (VI) and Ni (II) after treatment with Fe3O4-NH2 have been presented in Table −1 was 97.94 and 98.56 % for tap water and industrial wastewater, respectively. These measures were respectively obtained as 96.12 and 97.24 % for Ni (II) at the concentration of 1.0 mg L−1. This implies the excellent potential of Fe3O4-NH2 in water and wastewater treatment.In this study, 1.0 and 5 mg L3O4-NH2 was prepared using a simple, cost-effective, and environmentally friendly method for the removal of Cr (VI) and Ni (II) ions from aqueous solution and was characterized by SEM, TEM, XRD, and VSM. The effects of controlling parameters, such as contact time, temperature, pH, Fe3O4-NH2 dose, and initial concentration of both heavy metals, were studied, as well. Based on the results, the Langmuir model fitted the isotherm data for both heavy metals and the maximum sorption capacity was 232.51 mg g−1 at pH = 3 for Cr (VI) and 222.12 mg g−1 at pH = 6 for Ni (II). Moreover, the adsorption kinetic data for Cr (VI) and Ni (II) were based on the assumption of a pseudo-second-order model and thermodynamic parameters showed that the adsorption process was endothermic, spontaneous, and entropy favored in nature. In addition, this nano-adsorbent was able to remove over 96 % of both heavy metals from tap water and industrial wastewater. The Fe3O4-NH2 could be regenerated with acid after adsorption and the adsorption capabilities only decreased with 6-7 % for both metal ions after five cycles. Overall, this study indicated that an amino-functionalized magnetic nano-adsorbent was promising for removal of Cr (VI) and Ni (II) ions in field application.In this study, FeA sensitive method was developed for removal of Cr (VI) and Ni (II) from aqueous solution.3O4 nanoparticles.In-lab synthesized magnetic nanoadsorbent was developed by grafting amino-groups onto the surfaces of FeThe adsorbent was characterized by Transmission Electron Microscopy (TEM), powder X-Ray Diffraction (XRD), Vibrating Sample Magnetometry (VSM), and Scanning Electron Microscope (SEM).The effects of pH, initial concentrations of Ni (II) and Cr (VI), adsorption kinetics, thermodynamics, and adsorption isotherm were studied.SEM, scanning electron microscope; TEM, transmission electron microscopy; VSM, vibrating sample magnetometry; XRD, powder X-Ray diffraction


The substitution of a Br substituent by an I atom modifies the network of halogen bonds, and gives rise to the formation of non-classical Br 6H2Br3NO2 and C6H2Br2INO2, crystallize in triclinic and monoclinic cells, respectively, with two molecules per asymmetric unit in each case. The asymmetric unit of the tribromo compound features a polarized Brδ+⋯Brδ- intermolecular halogen bond. After substitution of the Br atom in the para position with respect to the nitro group, the network of X⋯X halogen contacts is reorganized. Two intermolecular polarized halogen bonds are then observed, which present the uncommon polarization Brδ+⋯Iδ-: the more electronegative site (Br) behaves as a donor and the less electronegative site (I) as an acceptor for the charge transfer.The title trihalogenated nitrobenzene derivatives, C X⋯B, shares many aspects with classical hydrogen bonds, but is more directional. On the other hand, halogen contacts X⋯X are more difficult to conceptualize , while Bhar et al. used a non-reduced cell. Moreover, the asymmetric unit content was defined in order to emphasize the strongest Br⋯Br bond in (I)P21/c.Both compounds crystallize with two molecules in the asymmetric unit, but in different space groups. The tribromo derivative, I, Fig. 1, is a PT = 100 K; Reddy et al., 2006T = 100 K; Brezgunova et al., 2012T = 100 K; Ghosh et al., 2007T = 223 K, Novak & Li, 2007The C—halogen bond lengths are as expected. In (I)Acta E article about 1,2,3-triiodobenzene  and 1(2)° for each independent molecule. For (II)2 groups is more significant, with dihedral angles of 10 (1) and 7(1)°. This near planar conformation is identical to that observed for 1,2,3-trichloro-5-nitrobenzene °; Stein et al., 2011ortho substituents. For 1,2,3-halogenated-5-nitrobenzenes such as (I)The 5-nitro substituent is almost conjugated with the benzene nucleus in (I)et al., 2006θ1 = C2—Br2⋯Br11 and θ2 = C11—Br11⋯Br2, which should be close to 180 and 90°, respectively. For (I)θ1 = 165.2 (5)° and θ2 = 82.3 (5)°. The crystal packing thus polarizes the involved halogen atoms, forming the halogen bond Br2δ+⋯Br11δ-. This dimolecular polar unit is connected via inversion centers to neighboring units in the cell, forming C—H⋯Br hydrogen bonds, and O⋯Br contacts. This packing motif induces secondary halogen⋯halogen contacts, which are clearly unpolarized. These type-I interactions are characterized by angles θ1 ≃ θ2 e.g. Bhar et al., 1995et al., 1974e.g. Olaru et al., 2014et al., 1996et al., 2010et al., 2012et al., 2008et al., 2012The current release of the CSD Synthesis of (I). A solution of 2,6-dibromo-4-nitroaniline  in acetic acid (3 ml) was cooled to 278 K, and concentrated H2SO4 (7 ml) was carefully added under stirring. While ensuring that the temperature was still below 278 K, NaNO2  was added in one batch. The reaction was stirred at this temperature for 2 h to afford the diazonium salt. An aqueous solution (17.67 ml) of CuBr  and 47% HBr (17.67 ml) was warmed to 343 K, and the diazotization solution previously prepared was added in one batch with stirring. The mixture was kept at 343 K for 1 h, and then left to cool overnight. The reaction was neutralized with NaOH and extracted with CH2Cl2 (3 × 30 ml). The resulting solution was concentrated under vacuum and the crude material was purified by flash chromatography  to give (I)−1): 3090 (Ar—H); 1583 (C=C); 1526, 1342 (N=O); 738 (C—Br). 1H-NMR : δ 8.43 . 13C-NMR : δ 146.8, 135.7, 127.0, 126.9, 126.8. EIMS m/z: [M+] 357 (34), [M++2] 359 (7), [M++4] 361 (100), [M++6] 363 (36) [M+-NO2] 311 (12).Synthesis of (II). A solution of 2,6-dibromo-4-nitroaniline  in acetic acid (3 ml) was cooled to 278 K in an ice-salt bath, and concentrated H2SO4 (3 ml) was carefully added under stirring. While ensuring that the temperature was still below 278 K, NaNO2  was added in one batch. The reaction was stirred at this temperature for 30 min to afford the diazonium salt. An aqueous solution (10 ml) of KI  was prepared, and the diazotization solution previously prepared was added in one batch. The mixture was then further stirred for 1 h. The reaction was neutralized with NaOH, extracted with CH2Cl2 (3 × 30 ml), and concentrated under vacuum. The crude material was purified by flash chromatography  to give (II)2Cl2 solution . m.p. 415–417 K. IR : 3010 (Ar—H); 1620, 1516 (C=C); 1336 (N=O). 1H-NMR : δ 8.38 . 13C-NMR : δ 146.1, 142.4, 127.4, 124.1. EIMS m/z: [M+] 405 (42), [M++2] 407 (100), [M++4] 409 (48).DIFABS on the complete isotropic model  = 1.2Ueq(carrier atom).Crystal data, data collection and structure refinement details for (I)10.1107/S2056989015013377/hb7459sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989015013377/hb7459Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015013377/hb7459IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989015013377/hb7459Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015013377/hb7459IIsup5.cmlSupporting information file. DOI: 1412444, 1412445CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A three-dimensional hydrogen-bonded network is observed which can be topologically simplified into a uninodal 5-connected {4 4H7N2+·C6H2N3O7−, the phenolic proton of the starting picric acid has been transferred to the imidazole N atom. The nitro groups are twisted away from the benzene ring plane, making dihedral angles of 12.8 (2), 9.2 (4) and 29.3 (2)°. In the crystal, the component ions are linked into chains along [010] via N—H⋯O and bifurcated N—H⋯ hydrogen bonds. These chains are further linked by weak C—H⋯O hydrogen bonds into a three-dimensional network. The complex three-dimensional network can be topologically simplified into a 4-connected uninodal net with the point symbol {4.85}.In the title molecular salt, C This is mainly due to the electron-withdrawing effect of the three nitro groups attached to the aromatic π system, delocalizing electron density on the phenolate oxygen atom over the π system. The three nitro groups, N1/O2/O3, N2/O4/O5 and N3/O6/O6, are twisted away from the benzene ring plane, making dihedral angles of 12.8 (2), 9.2 (4) and 29.3 (2)°, respectively. In the 4-methylimidazolium cation, the C9—N4 [1.321 (3) Å] and C9—N5 [1.304 (3) Å] bond lengths are similar to each other due to the delocalizing effect; this is in contrast to the un-protonated 4-methylimidazole molecule in the co-crystal of 8-hydroxyquinoline and 5-methyl-1H-imidazole . H atoms bonded to N atoms were found in Fourier difference maps; N—H distances were refined freely with Uiso(H) = 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901600712X/lh5809sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S205698901600712X/lh5809Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901600712X/lh5809Isup3.cmlSupporting information file. DOI: 1476728CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The dihedral angle between the phenyl ring and the benzimidazole ring system is significantly smaller in one of the compounds, viz. 73.42 (10) compared to 83.07 (17)°.In two benzoimidazo. In the crystal of (II), there are no significant intermolecular interactions present.The title compounds, C Here the methyl carboxylate group (C9/O2/O3/C10), planar to within 0.003 (2) Å, is inclined to the phenyl ring and the benzimidazole ring system by 53.04 (12) and 60.22 (11)°, respectively. These angles are also very different to those observed in compound (I)viz 33.78 (16) and 87.56 (14)°, respectively.The principle difference in the two compounds concerns the orientation of the phenyl ring (C15–C20) with respect to the benzimidazole ring system . In (II)a-axis direction n Table 1.In the crystal of (II)H,3H-benzimidazole -3-phenylacrylate (1.0 mol) and (1H-benzo[d]imidazole-2-yl)methanol (1.1 mol) for (I)H-benzo[d]imidazole-2-yl)ethanol (1.1 mol) for (II)2CO3 (1 mol) in CH3CN (10 ml) was stirred for 8 h. After completion of the reactions, monitored by TLC, the solvents were evaporated under reduced pressure. The residues were diluted with ethyl acetate then washed with brine and water. The organic layers were separated and the residues were subjected to column chromatography using ethyl acetate and hexane (2:8) as eluent. The products were dissolved in chloroform and heated for 2 min. The resulting solutions were subjected to crystallization by slow evaporation of the solvent for 48 h resulting in the formation of colourless block-like crystals of compounds (I)A mixture of Crystal data, data collection and structure refinement details are summarized in Table 2Uiso(H) = .2Ueq(C) or 1.5Ueq(Cmethyl). In both compunds, the C-bound H atoms were positioned geometrically and allowed to ride on their parent atoms: C—H = 0.93–0.98 Å with 10.1107/S1600536814021655/su2789sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S1600536814021655/su2789Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536814021655/su2789IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S1600536814021655/su2789Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S1600536814021655/su2789IIsup5.cmlSupporting information file. DOI: 1027182, 1027183CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H,4H-1,2,4-triazolium pyrazine-2-carboxylate was isolated by reacting 5-amino-1H-1,2,4-triazole-3-carboxylic acid and pyrazine-2,3-dicarboxylic acid in the presence of silver nitrate and 1-butyl-3-methylimidazolium bromide in ionothermal conditions.The salt 3-amino-2 H,4H-1,2,4-triazolium cation and the pyrazine-2-carboxylate anion in the title salt, C2H5N4+·C5H3N2O2−, were formed by an unexpected decarboxylation reaction, from 5-amino-1H-1,2,4-triazole-3-carboxylic acid and pyrazine-2,3-dicarboxylic acid, respectively. The dihedral angle between the pyrazine ring (r.m.s. deviation = 0.008 Å) and the carboxylate group in the anion is 3.7 (3)°. The extended structure of the salt contains a supramolecular zigzag tape in which cations and anions are engaged in strong and highly directional N—H⋯N,O hydrogen bonds, forming R22(8) and R22(9) graph-set motifs. The packing between the tapes is mediated by π–π stacking interactions between the triazole and pyrazine rings.Both the 3-amino-2 In the presence of AgNO3 the obtained product revealed, however, to be an unexpected organic salt -1,2,4-triazolium cation and the pyrazine-2-carboxylato anion.A remarkable feature of ionothermal synthesis is the fact that ionic liquids (ILs) can act simultaneously as sustainable solvents and structure-directing agents . This has been widely demonstrated by their potential in the discovery of unprecedented crystalline materials +] and pyrazine-2-carboxylate [(C5H3N2O2)−]. The asymmetric unit is composed of one of each of these moieties, as depicted in both the chemical diagram and in Fig. 1The title compound is a product of decomposition of the HD—H⋯A) interaction angles being above 165°. These supramolecular contacts are the main driving force which mediate the crystal packing features of the title compound. Indeed, the donation of hydrogen atoms from the cation to the carboxylate group of an adjacent anion (N6—H6B⋯O2 and N5—H5⋯O1) forms the known structurally robust et al., 1999Database survey below). Two other interactions, N6—H6A⋯N1 (dashed aqua lines) and N4—H4A⋯O2, describe a second The cation present in the title compound is rich in groups capable of forming strong N—H⋯N,O hydrogen-bonding interactions , pyrazine-2,3-dicarboxylic acid , 1-methylimidazole (99%+ purity), 1-bromobutane (99% purity) and AgNO3 (99%+ purity) were purchased from Sigma–Aldrich and were used as received without further purification. 1-Butyl-3-methylimidazolium bromide ([BMI]Br) was prepared according to the literature method .5-Amino-13 , H2atrc  and H2Pzdc  were mixed with 0.49 g of [BMI]Br and 0.3 mL of distilled water in a ca 25 mL Teflon-lined stainless-steel reaction vessel. The resulting mixture was heated to 383 K for 7 days. The vessel was then allowed to cool to ambient temperature at a rate of ca 1 K h−1. Small colourless crystals of the title compound were directly isolated from the vessel contents.AgNOUeq of the carbon atom to which they are attached. Hydrogen atoms associated with nitrogen atoms were located directly from difference Fourier maps and were included in the model with the N—H and H⋯H (only for the –NH2 groups) distances restrained to 0.90 (1) and 1.55 (1) Å, respectively, in order to ensure a chemically reasonable environment for these groups. These hydrogen atoms were modelled with the isotropic thermal displacement parameters fixed at 1.5Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901501172X/hb7446sup1.cifCrystal structure: contains datablock(s) I, New_Global_Publ_Block. DOI: 10.1107/S205698901501172X/hb7446Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901501172X/hb7446Isup3.cmlSupporting information file. DOI: 1407396CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Pathological inclusions containing aggregated, highly phosphorylated (at serine129) α-synuclein (αS pSer129) are characteristic of a group of neurodegenerative diseases termed synucleinopathies. Antibodies to the pSer129 epitope can be highly sensitive in detecting αS inclusions in human tissue and experimental models of synucleinopathies. However, the generation of extensively specific pSer129 antibodies has been problematic, in some cases leading to the misinterpretation of αS inclusion pathology. One common issue is cross-reactivity to the low molecular mass neurofilament subunit (NFL) phosphorylated at Ser473. Here, we generated a series of monoclonal antibodies to the pSer129 αS and pSer473 NFL epitopes. We determined the relative abilities of the known αS kinases, polo-like kinases (PLK) 1, 2 and 3 and casein kinase (CK) II in phosphorylating NFL and αS, while using this information to characterize the specificity of the new antibodies. NFL can be phosphorylated by PLK1, 2 and 3 at Ser473; however CKII shows the highest phosphorylation efficiency and specificity for this site. Conversely, PLK3 is the most efficient kinase at phosphorylating αS at Ser129, but there is overlay in the ability of these kinases to phosphorylate both epitopes. Antibody 4F8, generated to the pSer473 NFL epitope, was relatively specific for phosphorylated NFL, however it could uniquely cross-react with pSer129 αS when highly phosphorylated, further showing the structural similarity between these phospho-epitopes. All of the new pSer129 antibodies detected pathological αS inclusions in human brains and mouse and cultured cell experimental models of induced synucleinopathies. Several of these pSer129 αS antibodies reacted with the pSer473 NFL epitope, but 2 clones (LS3-2C2 and LS4-2G12) did not. However, LS3-2C2 demonstrated cross-reactivity with other proteins. Our findings further demonstrate the difficulties in generating specific pSer129 αS antibodies, but highlights that the use of multiple antibodies, such as those generated here, can provide a sensitive and accurate assessment of αS pathology.The online version of this article (doi:10.1186/s40478-016-0357-9) contains supplementary material, which is available to authorized users. Synucleinopathies are a group of neurodegenerative diseases that are characterized by the presence of proteinaceous inclusions containing aggregated α-synuclein (αS) –4. TheseGiven the extensive use of pSer129 αS antibodies to assess for the presence of pathological αS inclusions, it is important to generate and validate the specificity of these reagents. Indeed many pSer129 αS antibodies can cross-react with additional phosphorylated proteins and it has been difficult to generate antibodies that are highly specific , 18. ForHerein, we have generated a series of novel monoclonal pSer129 antibodies and characterized the relative preferential specificity of casein kinase (CK) II and polo-like kinases (PLK) 1, 2 and 3 to phosphorylate Ser129 in αS and Ser473 in NFL. Most of these new antibodies demonstrated some variable cross-reactivity with the pSer473 NFL epitope. However, we also determined that the previously generated antibody EP1536Y and the new antibodies LS4-2G12 and LC3-2C2 did not react with phosphorylated NFL, although EP1536Y and LC3-2C2 had a tendency to non-specifically react with cellular nuclei. We demonstrated that the new pSer129 αS antibodies could be used to detect pathological inclusions in patients with synucleinopathies, as well as in induced mouse and cell culture models of αS inclusion formation. Collectively, these resources will be highly valuable tools for the field to accurately assess and monitor αS inclusion formation.All procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals and were approved by the University of Florida Institutional Animal Care and Use Committee. BALB/c mice and αS null (αS KO) mice  were obtMouse anti-NFL antibody NR4 was obtained from Sigma-Aldrich . Anti-pSer129 αS rabbit monoclonal antibody EP1536Y was obtained from Abcam . Anti-human αS mouse monoclonal antibody Syn 204 was previously described . pSer129st injection; Sigma Aldrich, St. Louis, MO) or Freunds incomplete adjuvant  and vortexing for 15 mins until emulsified. Female BALB/c mice  were injected subcutaneously. A second subcutaneous injection was administered 3 weeks later. Six weeks following the initial injection, mice were boosted with an intraperitoneal (IP) injection of 100 μg KLH-conjugated peptide in PBS. Three days later, mice were euthanized and spleens were harvested using aseptic technique.Peptides Table  designed2. Spleens were gently homogenized in 5 % FBS/Hank’s balanced salt solution  and centrifuged to pellet cells. The cell pellet was resuspended in red blood cell lysis buffer  and diluted with HBSS after one minute. The cells were then washed twice by centrifuging at 100 × g for 10 mins and resuspended in HBSS. Sp2/O-Ag14 cells were also washed twice with HBSS. Five million Sp2/O-Ag14 cells were added to 50 million spleen cells and, after centrifuging at 100 × g for 10 mins onto a culture dish, fusion was induced with 50 % polyethylene glycol 1450 . After washing with HBSS, cells were incubated in Sp2/O-Ag14 media at 37 °C with 8 % CO2 overnight. The next day, the cells were gently detached from the plate and distributed into 96 well plates with Sp2/O-Ag14 media/0.5 % hybridoma enhancing supplement /HAT selection supplement .Mouse myeloma  cells were maintained in high glucose (4.5 g/L) Dulbecco’s Modified Eagle Medium (DMEM) with 10 % NCTC 135 Media , 20 % hybridoma grade fetal bovine serum , 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM L-glutamine, 0.45 mM pyruvate, 1 mM oxaloacetate, and 0.2 U/ml insulin at 37 °C and 8 % COAll hybridoma clones were screened for reactivity to the injected peptide by enzyme-linked immunosorbent assay (ELISA). MaxiSorp plates  were coated with 1 μg/ml peptide in PBS and blocked with 5 % FBS/PBS. Media from the hybridomas were applied to plates, which were then incubated at room temperature for 3 h. Next, the plates were washed with PBS, and incubated with goat anti-mouse secondary antibody conjugated to horse radish peroxidase  for 1 h at room temperature. Then, plates were washed and TMB substrates  were applied until color changes were observed. Reactions were then quenched with 1 M HCl and absorbance was measured at 450 nm. Clones that were positive by ELISA were transferred to larger culture plates as needed. The positive clones were next screened by immunohistochemistry (IHC) of a human autopsy case with abundant αS pathology.Paraffin embedded tissue from αS transgenic and WT mice are described in Table Escherichia coli (E. coli) BL21 (DE3)/RIL  using the respective cDNA cloned into the bacterial expression plasmid pRK172, and purified as previously described [Recombinant WT human or mouse αS or human αS with serine 129 mutated to alanine (S129A) were expressed in escribed , 32. ProE. coli BL21 (DE3)/RIL , using the respective cDNA cloned in the bacterial expression plasmid pET-23d and purified as previously described [Recombinant WT mouse NFL or mouse NFL with serine 473 mutated to alanine (S473A) were expressed in escribed . Protein32P ATP  in manufacturer recommended buffers , 2 mM dithiothreitol (DTT), 0.01 % Brij 35; PLK1 buffer: 50 mM HEPES, pH7.5, 10 mM MgCl2, 2.5 mM DTT, 0.01 % Triton X-100; PLK2 buffer: 25 mM HEPES, pH7.5, 10 mM MgCl2, 5 mM MnCl2, 0.5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 0.5 mM Na3VO4, 5 mM β-glycerophosphate, 2.5 mM DTT, 0.01 % Triton X-100; PLK3 buffer: 25 mM Tris, pH7.5, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3VO4, 5 mM β-glycerophosphate, 2.5 mM DTT, 0.01 % Triton X-100). Reactions without protein for each kinase, and without kinase for each protein, were included as control reactions. Each reaction was performed in triplicate. Reactions were then absorbed onto P-81 phosphocellulose filters  and immersed in 85 mM phosphoric acid. After extensive washing in 85 mM phosphoric acid, the filters were rinsed with acetone, air dried and placed in scintillation vials with Econo-Safe Liquid Scintillation cocktail . Radioactivity (32P) incorporation was measured using an LS5000 TD liquid scintillation counter .To determine which kinases could phosphorylate αS and NFL, and to what extent, we performed radioactive kinase reactions. NFL and S473A NFL were dialyzed in Tris or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) overnight to remove the urea. Recombinant proteins  were incubated in 25 μl reactions at 30 °C for 8 h with 60 ng PLK2  or 20 ng CKII , PLK1 or PLK3 , 200 μM adenosine triphosphate (ATP) with γ-32P ATP. Sodium dodecyl sulfate (SDS) sample buffer was added to the reactions which were incubated for 10 mins at room temperature. Equimolar amounts of protein were resolved by SDS-PAGE and analyzed by immunoblot.CKII and PLK3 reactions for immunoblot antibody testing were performed as before, but without γ-Mouse nervous tissues were fractionated as described by Emmer et al. 2011 . BrieflyMice were humanely euthanized and the brain and spinal cord were harvested. The cortex and brainstem/spinal cord were dissected, placed in separate tubes and lysed with 2 % SDS/50 mM Tris, pH 7.5. Samples were sonicated using a probe sonicator until homogenous and incubated for 10 mins at 100 °C. Protein concentrations were determined by BCA assay using BSA as a standard. SDS sample buffer was added and equal amounts of protein (5 μg) were resolved by SDS-PAGE and analyzed by immunoblot.Protein samples were resolved by electrophoresis on 8, 10 or 13 % polyacrylamide gels as indicated, then electrophoretically transferred to nitrocellulose membranes. Membranes were blocked with 5 % milk/Tris-buffered saline (TBS) then incubated overnight at 4 °C with primary antibodies diluted in 5 % milk/TBS (non-phospho antibodies) or 5 % BSA/TBS (phospho antibodies). Following washing, blots were incubated with HRP conjugated goat anti-mouse or donkey anti-rabbit secondary antibodies  diluted in 5 % milk/TBS for 1 h. Following washing, protein bands were visualized using Western Lightning-Plus ECL reagents  and images were captured using the GeneGnome XRQ system and GeneTools software .2. Cells were maintained in the Neurobasal-A growth media without FBS at 37 °C in a humidified 5 % CO2 chamber. The cultures were maintained for 6 days followed by another 8 days without or with 20 μg/ml water bath sonicated mouse αS fibrils prepared as previously described [Primary cultures (embryonic) were prepared from E16-E18 C3HBL/6 or αS null mouse brains. Cerebral cortices were dissected from E16-E18 mouse brains and were dissociated in 2 mg/ml papain  and 50 μg/ml DNAase I  in sterile HBSS at 37 °C for 20 mins. They were washed three times in sterile HBSS to inactivate the papain and switched to 1 % FBS in Neurobasal-A growth media , which includes 1 % GlutaMax Supplement , B-27 supplement, 100 U/ml penicillin and 100 μg/ml streptomycin. The tissue mixture was triturated three times using a 5 ml pipette followed by a Pasteur pipette, and strained through a 70 μm nylon cell strainer. The cell mixture was then centrifuged at 1300 × g for 3 mins, and resuspended in fresh Neurobasal-A media. They were then plated on Nunc Lab-Tek II CC2 chamber slides  at around 100,000-200,000 cells/cmescribed , 35.For double immunofluorescence analysis, cells were washed with PBS and fixed with 4 % paraformaldehyde/PBS. Following PBS washes, cells were blocked with 5 % FBS/PBS/0.1 % Triton X-100 for 30 mins. Cultures were stained with primary antibodies followed by Alexa-fluor 488 and 594 conjugated secondary antibodies . Nuclei were counterstained with 4′,6-diamidino-2-phenylindole , and coverslips were mounted using Fluoromount-G . Immunofluorescence images were captured with an Olympus BX51 fluorescence microscope mounted with a DP71 digital camera .CKII was previously reported as a major kinase responsible for the phosphorylation of αS, more specifically at Ser129 , 36, 37,To provide more reagents to study the contribution of aberrant accumulation and/or distributions of αS and NFL in neurodegenerative diseases, we generated novel monoclonal antibodies against the pSer473 epitope in NFL and the pSer129 epitope in αS. For the pSer473 NFL epitope we used a synthetic peptide with phosphorylated serine and the adjacent 7 amino acids residues on either side in the human NFL sequence (Table For the pSer129 αS epitope we performed multiple attempts to produce αS antibodies that were 1) phospho-specific and 2) relatively specific in detecting Lewy pathology. For our most successful approach, the mice were first immunized with the pSer129long peptide (αS residues 121–137) then 3 weeks later with the pSer129short peptide , cingulate cortex , we confirmed that all the antibodies could be used to stain tissue preserved with both types of fixatives -N,N,N’,N’-tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GCI, glial cytoplasmic inclusion; GRK, G-protein coupled receptor kinase; HBSS, Hank’s balanced salt solution; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HRP, horse radish peroxidase; HS, high salt; HS/T, high salt/Triton X-100; IHC, immunohistochemistry; IP, intraperitoneal; KO, null (knock-out); LB, Lewy body; M83-I, motor impaired M83 mouse; MSA, multiple system atrophy; NCI, neuronal cytoplasmic inclusion; NFID, neurofilament inclusion disease; NFL, low molecular mass neurofilament subunit; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PD, Parkinson’s disease; PEG, polyethylene glycol; PLK, polo-like kinase; RIPA, radioimmunoprecipitation assay; pSer, phosphorylated serine; SDS, sodium dodecyl sulfate; SDS/U, sodim dodecyl sulfate/urea; TBS, Tris-buffered saline; WT, wild-typeαS, α-synuclein; αS fib, α-synuclein fibrils; ATP, adenosine triphosphate; BCA, bicinchoninic acid; BSA, bovine serum albumin; BS/SC, brainstem/spinal cord; CK, casein kinase; Ct, control; DAB, 3, 3′diaminobenzidine; DAPI, 4′,6-diamidino-2-phenylindole; DLB, dementia with Lewy bodies; DMEM, Dulbecco’s Modified Eagle Medium; DTT, dithiothreitol;


The crystal packing is dominated by an extensive network of N—H⋯O hydrogen bonds, resulting in a high density of 1.795 Mg m 2NC(=O)NHC(NH2)2]+− (= guanylurea dipicrylamide), was prepared as dark-red block-like crystals in 70% yield by salt-metathesis reaction between guanylurea sulfate and sodium dipicrylamide. In the solid state, the new compound builds up an array of mutually linked guanylurea cations and dipicrylamide anions. The crystal packing is dominated by an extensive network of N—H⋯O hydrogen bonds, resulting in a high density of 1.795 Mg m−3, which makes the title compound a potential secondary explosive.The title compound, 1-carbamoylguanidinium bisamide [H Several significant advantages such as high heats of combustion, high propulsive power, high specific impulse, as well as smokeless combustion make them highly useful as propellants, explosives, and pyrotechnics NHC(NH2)2]2. The guanylurea cation has frequently been reported to be a useful component in energetic nitrogen-rich salts, e.g. guanylurea dinitramide NHC(NH2)2]2SO4  NH2] = ca 6.3–7.1 p.p.m., δ[NHC(NH2)2] = ca 3.3–5.8 p.p.m.). In contrast, interpretation of the 13C NMR spectrum was straightforward. In perfect agreement with the 13C NMR data of previously reported ammonium and azolium DPA salts 2). IR bands in the range of 3200–3400 cm−1 were characteristic for the N—H valence vibrations in the guanylurea cation. A strong carbonyl band was observed at 1632 cm−1, whereas the band at 1532 cm−1 is characteristic for the nitro groups.In the course of our ongoing studies on the crystal structures of energetic compounds (Deblitz P− anion is 131.66 (10)°, with C—N bond lengths of 1.3021 (15) (C1—N1) and 1.3403 (15) Å (C7—N1). These values are somewhat shorter than the C—N bond lengths in free dipicrylamine 2 is nearly trigonal-planar with N—C—N angles between 117.52 (11) and 121.48 (11)° and C—N distances in the very narrow range of 1.3064 (15)–1.3158 (15) Å. Overall, the structural parameters of the cation in the title compound do not differ significantly from those in 1-carbamoylguanidinium sulfate, [H2NC(=O)NHC(NH2)2]2SO4 2 engage in four N—H⋯O hydrogen bonds to three different nitro groups of the DPA− anion. The calculated density of 1.795 Mg m−3 is not only higher than the densities reported for other DPA-based salts (1.69–1.78 Mg m−3), but also much higher than the density of TNT (1.65 Mg m−3)  of guanylurea dipicrylamide have not been tested, but recent findings have shown that the impact sensitivities of various ammonium and azolium dipicrylamides are in the range of that of the secondary explosive TNT : C 31.06, H 2.05, N, 28.46; found: C 31.87, H 2.27, N 28.10%. IR (KBr pellet): νmax 3403 (vs), 2171 (w), 1632 (vs), 1532 (s), 1402 (vs), 1270 (s), 1129 (m), 924 (w), 878 (m), 840 (w), 773 (w), 701 (m), 622 (m), 452 (m). 1H NMR : δ = 8.78 3), ca 6.3–7.1 , ca 3.3–5.8  p.p.m. 13C NMR : δ = 157.4 (C=O); 155.5 [NHC(NH2)2]; 143.4, 139.5, 132.6, 125.1 [C6H2(NO2)3] p.p.m.Preparation of guanylurea dipicrylamide: To a suspension of dipicrylamine  in 10 ml water were added two pellets of NaOH to give a dark red solution of sodium dipicrylamide; 0.23 g (1.5 mmol) of guanylurea sulfate were added as solid, and the mixture was allowed to stand undisturbed at room temperature. After 10 d, 0.87 g (70%) dark-red crystals of the title compound had formed, which were isolated by filtration and dried in air. Analysis calculated for CCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S1600536814017164/zl2596sup1.cifCrystal structure: contains datablock(s) I, New_Global_Publ_Block. DOI: 10.1107/S1600536814017164/zl2596Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814017164/zl2596Isup3.cmlSupporting information file. DOI: 1015895CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Eleven new human polyomaviruses (HPyVs) have been identified in the last decade. Serological studies show that these novel HPyVs sub-clinically infect humans at an early age. The routes of infection, entry pathways, and cell tropism of new HPyVs remain unknown. VP1 proteins of polyomaviruses can assembly into virus-like particles (VLPs). As cell culturing systems for HPyV are currently not available, VP1-derived VLPs may be useful tools in basic research and biotechnological applications.Recombinant VP1-derived VLPs from 11 newly identified HPyVs were efficiently expressed in yeast. VP1 proteins derived from Merkel cell polyomavirus (MCPyV), trichodysplasia spinulosa-associated polyomavirus (TSPyV), and New Jersey polyomavirus (NJPyV) self-assembled into homogeneous similarly-sized VLPs. Karolinska Institutet polyomavirus (KIPyV), HPyV7, HPyV9, HPyV10, and St. Louis polyomavirus (STLPyV) VP1 proteins formed VLPs that varied in size with diameters ranging from 20 to 60 nm. Smaller-sized VLPs (25–35 nm in diameter) predominated in preparations from Washington University polyomavirus (WUPyV) and HPyV6. Attempts to express recombinant HPyV12 VP1-derived VLPs in yeast indicate that translation of VP1 might start at the second of two potential translation initiation sites in the VP1-encoding open reading frame (ORF). This translation resulted in a 364-amino acid-long VP1 protein, which efficiently self-assembled into typical PyV VLPs. MCPyV-, KIPyV-, TSPyV-, HPyV9-, HPyV10-, and HPyV12-derived VLPs showed hemagglutination (HA) assay activity in guinea pig erythrocytes, whereas WUPyV-, HPyV6-, HPyV7-, STLPyV- and NJPyV-derived VP1 VLPs did not.The yeast expression system was successfully utilized for high-throughput production of recombinant VP1-derived VLPs from 11 newly identified HPyVs. HPyV12 VP1-derived VLPs were generated from the second of two potential translation initiation sites in the VP1-encoding ORF. Recombinant VLPs produced in yeast originated from different HPyVs demonstrated distinct HA activities and may be useful in virus diagnostics, capsid structure studies, or investigation of entry pathways and cell tropism of HPyVs until cell culture systems for new HPyVs are developed. Orthopolyomavirus and Wukipolyomavirus) and avian (Avipolyomavirus) species , according to the native virus gene sequence with one nucleotide changed to remove a restriction endonuclease XbaI recognition site, and 2) HPyV12 VP1s380 with a S. cerevisiae codon-optimized gene sequence [GenBank: KP293751]. Shorter 364-aa-long versions of the HPyV12 VP1 protein with native (HPyV12 VP1n364) and codon-optimized sequences (HPyV12 VP1s364) were amplified from HPyV12 VP1n380 and HPyV12 VP1s380 using Phusion High-Fidelity DNA Polymerase and the primers: HPy12-VP1ATG2-NX: ctctagaatggcacccaagaggaaaaccacctg; HPy12-VP1stop-NX: gtctagattatggaactggtgttatttcttgtc; HPy12-VP1-ATG2-SX: ctctagaatggcacctaaaaggaagaccacatg; HPy12-VP1stopSX: gtctagattatggaactggagtaatttc.Two versions of the HPyV12 VP1 protein, consisting of a 380-aa chain, were commercially synthesized by GenScript: 1) HPyV12 VP1nEscherichia coli DH5α cells.All VP1-encoding DNA fragments were sub-cloned into the XbaI site of the yeast expression vector pFX7 for target protein expression . Recombi380, pFX7-HPyV12-VP1s380, pFX7-HPyV12-VP1n364, pFX7-HPyV12-VP1s364, and pFX7-NJPyV-VP1 were transformed into the S. cerevisiae strain, AH22-214 . The growing conditions for yeast transformants harboring plasmids with VP1-encoding genes from different HPyVs and conditions for induction of VP1 synthesis were similar to that previously described [2, 0.25 M L-Arginine, 0.001 % Triton X-100, 10 mM Tris/HCl, pH 7.2), lysed by vortexing with an equal volume of glass beads for 7 min, and then cooled on ice for 1 min between each vortexing step. A 15–30-μL aliquot of each yeast lysate and the supernatant obtained after centrifugation of each yeast lysate were prepared for analysis by mixing with the SDS-PAGE sample buffer  and then boiling for 5 min. Prepared samples were then subjected to SDS-PAGE and run in SDS-Tris-glycine buffer. The resultant gel was stained with Coomassie brilliant blue  for protein band visualization.The vector pFX7, without any insert or constructed plasmids, pFX7-KIPyV-VP1, pFX7-WUPyV-VP1, pFX7-MCPyV-VP1, pFX7-HPyV6-VP1, pFX7-HPyV7-VP1, pFX7-TSPyV-VP1, pFX7-HPyV9-VP1, pFX7-HPyV10-VP1, pFX7-STPyV-VP1, pFX7-HPyV12-VP1nescribed . Briefly2, 0.001 % 0.25 M L-Arginine, and Trition X-100, in 10 mM Tris/HCl-buffer, pH 7.2) with 2 mM PMSF and EDTA-free Complete Protease Inhibitor Cocktail tablets , and then were homogenized with glass beads using Bead-Beater GB26 . The supernatant collected after centrifugation of yeast lysate was loaded onto a 20–69 % sucrose gradient in DB150 buffer and then centrifuged at 100,000 × g  overnight at 4 °C. Thereafter, 2-mL fractions were collected and samples from each fraction were analyzed using Coomassie brilliant blue-stained SDS-PAGE. Fractions containing VP1 were pooled, diluted in DB150 buffer, and after a 0.5 h incubation step with DNAse-free RNAse , VP1-derived VLPs were concentrated by ultracentrifugation at 100,000 × g for 4 h at 4 °C. Afterwards, pellets containing VP1 were dissolved in DB150 buffer and subjected to ultracentrifugation on a CsCl gradient with densities ranging from 1.23 to 1.46 g mL−1 at 100,000 × g overnight at 4 °C. Fractions were collected and analyzed, as described above. After pooling and diluting purified VP1-containing fractions in DB150 buffer, VP1-derived VLPs were precipitated by ultracentrifugation for 4 h at 100,000 × g. Pellets with VP1-derived VLPs were dissolved in PBS, dialyzed against PBS overnight, aliquoted, and then lyophilized or stored in PBS in 50 % glycerol at −20 °C. JCPyV, BKPyV, SV40, and HaPyV VP1-derived VLPs were prepared as previously described [Recombinant VP1 protein purification was carried out as previously described . Brieflyescribed .To confirm VLP assembly of recombinant VP1 proteins generated in yeast, a sample of VP1 purified by ultracentrifugation was placed on 300-mesh carbon-coated palladium grid. VP1-derived VLPs adhering to the grid were negatively stained with 2 % aqueous uranyl acetate solution and examined with a Morgagni 268 electron microscope .2O2 .Samples of purified recombinant VP1 proteins derived from all HPyVs and HaPyV were mixed with the SDS-PAGE sample buffer , boiled for 5 min, applied to a SDS-PAGE and run in SDS-Tris-glycine buffer. Protein bands in SDS-PAGE were electro-transferred to Immobilon P membrane  or visualized by staining with Coomassie brilliant blue . The membranes were blocked with 5 % milk in TTBS  and incubated overnight in the relevant antibody solutions at room temperature. Anti-JCPyV, −MCPyV, and -WUPyV VP1 mouse sera (raised against the respective VP1 protein) and anti-HaPyV VP1 rabbit serum  were dil−1 to 0.025 μg mL−1) in 25 μL PBS and mixed with 25 μL of a 1 % (v/v) guinea pig erythrocyte suspension in PBS. HA activity was recorded after a 3 h incubation at 4 °C. The HA titer was defined as the reciprocal of the highest antigen dilution clearly showing HA.VLPs were subjected to a HA assay using guinea pig erythrocytes in a U-shaped microtiter plate. VLPs of each purified VP1 protein were serially diluted (50 μg mL


Glucagon like peptide-1 (GLP-1), an incretin hormone, regulates glucose metabolism by inducing insulin secretion and suppressing glucagon secretion. The aim of the study is to assess the levels of fasting and post-prandial GLP-1 and their risk for T2DM. A case control study was conducted at the diabetes clinic Sanglah Hospital Denpasar Bali, involving 40 subjects who were native Indonesian citizens and 18–70 years of age. Twenty subjects were allocated as the case group (subjects with T2DM) and 20 subjects were allocated as the control group . Both fasting intact GLP-1 (FGLP-1) and 60 minutes post-75 gram glucose loading intact GLP-1 (1hGLP-1) levels were measured.vs. 2.87 ± 0.67 pg/L, p < 0.01; and 2.49 ± 0.60 vs. 3.42 ± 0.85 pg/L, p = 0.02; respectively). Low levels of FGLP-1  and 1hGLP-1 , with no response after glucose loading (∆GLP-1), were a significant risk for T2DM. According to the ∆GLP-1, there was a tendency of decreasing response of GLP-1 after glucose loading among subjects with T2DM (∆ = 0.43 pg/L) compared to subjects with NGT (∆ = 0.55 pg/L).Both fasting and post-prandial GLP-1 levels were significantly lower in subjects with T2DM than those with NGT (2.06 ± 0.43 From this study it can be concluded that levels of intact GLP-1 are an important risk factor for T2DM in the Indonesian population. Type 2 diabetes mellitus (T2DM) is a metabolic disease. It is a result of a combination of genetic predisposition, lifestyle and environment that are considered to be involved in its pathogenesis. Recently it has been widely known that changes of behavior, ways of life and the environment are predominant risk factors for T2DM . The preThe concept that gut endocrine stimulates pancreatic secretion was first hypothesized in 1902. It was considered a big leap in the understanding of incretins that function as a regulator of blood glucose through regulation of insulin and glucagon secretion . Two gutIn Indonesia, no data on GLP-1 levels are available both in normal subjects and subjects with T2DM. Thus, this study was conducted to assess the GLP-1 levels in fasting and after glucose loading states and the response of GLP-1 after glucose loading; and to prove that low GLP-1 levels was a risk factor of T2DM in the Indonesian population.A case–control study was conducted at the Diabetes Clinic Sanglah Hospital, Denpasar, Bali, Indonesia, during the period of February to April 2014. Twenty cases (subjects with T2DM) and 20 subjects with normal glucose tolerance (NGT), aged 18–70 years, were enrolled in the study as the control group. All subjects were native Indonesian citizens. The diagnosis of diabetes was confirmed based on the criteria of ADA (2014) [χ2 test and prevalence risk or odds ratio [OR]), with significant value confirmed at P < 0.05.Statistical tests used to analyze the data in the study included a descriptive presentation, a Mann–Whitney test, cross-tab (P < 0.01).The mean of FGLP-1, 1hGLP-1, and ∆GLP-1 are seen in Table vs. 2.87 ± 0.67 pg/L, P < 0.01; and 2.49 ± 0.60 vs. 3.42 ± 0.85 pg/L, P = 0.02; respectively).The mean age of the subjects was 56.85 ± 5.71 years . Body mass index in subjects with T2DM was higher than subjects with NGT, 28.94 ± 1.96 and 24.79 ± 3.07, respectively . Low levels of FGLP-1  and 1hGLP-1  were significantly a risk factor for T2DM, but not for ∆GLP-1 . Velasquez-Mieyer et al. in 2008 reported that African-Americans exhibited lower GLP-1active levels at 15 min after OGTT than Caucasians  [active in African-Americans was lower than in Caucasians (3.31 ± 1.14 vs. 6.67 ± 3.84 pmol/L) but the difference was not statistically significant. As summarized by Seino et al. in 2010, the intact GLP-1 level was lower in Japanese than Caucasians [vs. 76.4 ± 4.47 pg/mL, P < 0.001) [This study revealed that the levels of GLP-1 in fasting and post-prandial states in subjects with T2DM were lower than in subjects with NGT (2.06 < 0.001) . From th< 0.001) , 13.Low levels of FGLP-1  and 1hGLP-1  seen in this study were significant risk factors for T2DM. Although the ∆GLP-1 was not a significant risk factor for T2DM, it revealed a trend of decreased response of intact GLP-1 after glucose loading among subjects with T2DM (0.43 pg/L) compared to subjects with NGT (0.55 pg/L). Two hours total GLP-1 increments (∆GLP-1) following OGTT reduced significantly in T2DM (4.26 ± 6.27 pmol/L) group compared to NGT (12.37 ± 10.05 pmol/L) and isolated IFG (10.37 ± 9.82 pmol/L) or IGT (9.79 ± 10.50 pmol/L) groups .Since there were differences in the type of GLP-1 , method and unit GLP-1 measurement used in several different studies, there has been difficulty in comparing the absolute value between this study and other studies. However, it can be concluded that there was similarity in the findings that the levels of FGLP-1 and response after meal or glucose loading of GLP-1 were reduced in subjects with T2DM compared to subjects with NGT.Both FGLP-1 and 1hGLP-1 levels were lower in subjects with T2DM than in subjects with NGT. Low level of GLP-1 was an important risk factor of T2DM.


Enantiopure imines synthesized starting from 2-naphthaldehyde and chiral liquid amines were characterized by X-ray diffraction. S)-(+)-2-{[(1-phenylethyl)imino]methyl}naphthalene, C19H17N, (1), (S)-(+)-2-({[(4-methylphenyl)ethyl]imino}methyl)naphthalene, C20H19N, (2), (R)-(−)-2-({[(4-methoxylphenyl)ethyl]imino}methyl)naphthalene, C20H19NO, (3), (R)-(−)-2-({[(4-fluorophenyl)ethyl]imino}methyl)naphthalene, C19H16FN, (4), (S)-(+)-2-({[(4-chlorophenyl)ethyl]imino}methyl)naphthalene, C19H16ClN, (5), (S)-(+)-2-({[(4-bromophenyl)ethyl]imino}methyl)naphthalene, C19H16BrN, (6), (S)-(+)-2-ethyl]imino}methyl)naphthalene, C23H19N, (7), (S)-(+)-2-{[(1-cyclohexylethyl)imino]methyl}naphthalene, C19H23N, (8), (S)-(−)-2-{[imino]methyl}naphthalene, C21H19N, (9), and (+)-2--2,6,6-trimethylbicyclo[3.1.1]hept-3-yl]imino}methyl}naphthalene, C21H25N, (10). The moiety provided by the amine generates conformational flexibility for these imines. In the crystals, no strong intermolecular contacts are observed, in spite of the presence of aromatic groups.Using a general solvent-free procedure for the synthesis of chiral Schiff bases, the following compounds were synthesized and their crystal structures determined: ( Such procedures may overcome, for example, the hydrolytic susceptibility of the formed imine, since water is eliminated as a gas if the Schiff condensation is exothermic. A recent work in this direction was published, which reports the preparation of 15 Schiff bases formed between 3-ethoxysalicylaldehyde and primary aromatic amines  and an UV laser (405 nm), respectively, for eight Schiff bases. These results will be reported elsewhere in due course, along with electrical conduction studies.P21 or P212121. The absolute configuration was determined from anomalous dispersion effects in two crystals, for chlorine and bromine compounds (5) and (6), confirming that the enantiopure amines used as starting materials transfer the chiral center to the formed imines, without inversion. The Flack parameters for these crystals converged to 0.02 (6) and −0.009 (6), respectively. For the other compounds, the absolute configuration was assumed from the synthesis. Two compounds crystallize with four molecules per asymmetric unit, (1)  , while other compounds are obtained with a single molecule in the asymmetric unit. All imines (1)–(8) bear a single chiral C atom (C12) presenting the S configuration, except for compounds (3) and (4), which are R isomers. The chiral center linking aromatic moieties in (1)–(7) induces a bent shape for these molecules, and the dihedral angle formed by these moieties may be close to 90°. For instance, in the case of compound (2)  Fig. 1 and (8) 2) Fig. 2, the nap1)–(6) are closely related –(6)  to −79.2 (6)° for −synclinal angle τ2. For the interplanar dihedral angle δ, the observed range is from 43.7 (3) to 81.04 (11)°. Interestingly, (2), (5) and (6) have very similar metrics, probably as a consequence of the similar steric volumes for CH3, Cl, or Br groups. Angles τ1, τ2 and δ for (7)  –(6) because C12 is not substituted by a phenyl ring for these compounds  and (10) –(8), because the chiral center bonded to the imine group belongs to a cyclic system, introducing a strong restriction to the conformational flexibility.Finally, compounds (0) Fig. 7 are stru1)–(8). For (9) and (10), only one single bond is involved in conformational flexibility, N1—C12.It is worth noting that in all molecular structures, the imine bond remains conjugated with the naphthyl group. Rotational motions are thus possible only around bonds N1—C12 and C12—C14 for (1), which crystallizes with four molecules in the asymmetric unit, the mean planes of naphthyl groups stacked along [100] are separated by more than 6.5 Å. A consequence of the lack of stabilizing intermolecular forces in these crystals is their quite low packing index, in the range 63.5 [for (8)] to 67.0% [for (10)], and the occurrence of asymmetric units containing multiple molecules in the case of (1) and (8). For these compounds, the free rotation for the phenyl (1) or cyclohexyl (8) groups accounts for Z′ = 4 asymmetric units. Moreover, (1) and (8) crystallize in the same space group, P21, with similar unit-cell parameters, and similar arrangements for the molecules in the crystal. In other words, the substitution of a planar phenyl group by a non-planar cyclohexyl group has little influence on the crystal structure.A common feature may be observed over the series of crystal structures: despite the presence of aromatic systems, the molecules are arranged in such a way that no π–π interactions are favored. As an example, in the case of (2), (3), (5) and (6), where the benzene ring is para-substituted by non-sterically demanding functional groups, CH3, OCH3, Cl and Br, respectively. These four compounds crystallize in space group P212121 with unit-cell volumes of ca 1600 Å3 (see Table 24), is not isomorphous to analoguous compounds bearing Cl (5) and Br (6). Again, this behavior may be related to the rotational freedom of the benzene ring, which modifies the molecular conformation stabilized in the solid state.The same kind of crystal structure similarity is observed for ] and 1-cyclohexylethylamine [as in (8)]. For others, only sporadic X-ray determinations are carried out, for example for salicylaldimines –(10) was performed under solvent-free conditions. Reactants were mixed for 3–5 min. using a mortar and pestle, yielding oily products that become solids standing on air for an additional 2–3 minutes. The reaction was monitored by TLC and 1H NMR, observing the disappearance of the aldehyde. Because of the direct interaction and because no excess of reagents were used, the products were obtained with no waste and no further purification processes were needed. In most cases the products were obtained in a sufficiently pure form, or a simple crystallization was enough, when necessary, and in our case, the obtained crude products were recrystallized from CH2Cl2, affording the corresponding pure Schiff bases (1)–(10) as crystals of good quality for X-ray studies.The reaction of optically pure primary amines with 2-naphthaldehyde to yield chiral Schiff bases (1)–(10) showed characteristic absorption bands in the 1635–1626 cm−1 range, due to the C=N stretching vibration, in agreement with reported values. In the 1H NMR spectra, the azomethine proton appears in the 8.33–8.51 p.p.m. range, while the imine bond is characterized in the 13C NMR spectra with the imine C signal in the 157.9–160.7 p.p.m. range. Full spectroscopic data are available in the supporting information.The IR spectra (1) and (8) were twinned by a twofold rotation, with the same twin law  converged towards rather disappointing refinements, as a result of packing issues and disordered parts that were not well resolved. For (8), two cyclohexyl rings, C14–C19 and C74–C79, were restrained to have the same bond lengths as those of ring C34–C39. Other structures were refined without restraints.Crystal data, data collection and structure refinement details are summarized in Table 21)–(9), H atoms were placed in idealized positions, with C—H bond lengths fixed at 0.93 (aromatic), 0.96 (methyl), 0.97 (methylene) or 0.98 Å (methine). For (10), collected at 150 K, these distances were fixed at 0.95, 0.98, 0.99 and 1.00 Å, respectively. In all compounds, isotropic displacement parameters for H atoms were calculated as Uiso(H) = xUeq(carrier atom) with x = 1.5 (methyl) or x = 1.2 .For  1, 2, 3, 4, 5, 6, 7, 8, 9, 10, global. DOI: 10.1107/S2056989016004692/hb75711sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989016004692/hb75712sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S2056989016004692/hb75713sup4.hklStructure factors: contains datablock(s) 3. DOI: 10.1107/S2056989016004692/hb75714sup5.hklStructure factors: contains datablock(s) 4. DOI: 10.1107/S2056989016004692/hb75715sup6.hklStructure factors: contains datablock(s) 5. DOI: 10.1107/S2056989016004692/hb75716sup7.hklStructure factors: contains datablock(s) 6. DOI: 10.1107/S2056989016004692/hb75717sup8.hklStructure factors: contains datablock(s) 7. DOI: 10.1107/S2056989016004692/hb75718sup9.hklStructure factors: contains datablock(s) 8. DOI: 10.1107/S2056989016004692/hb75719sup10.hklStructure factors: contains datablock(s) 9. DOI: 10.1107/S2056989016004692/hb757110sup11.hklStructure factors: contains datablock(s) 10. DOI: Click here for additional data file.10.1107/S2056989016004692/hb75711sup12.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016004692/hb75712sup13.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016004692/hb75713sup14.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016004692/hb75714sup15.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016004692/hb75715sup16.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016004692/hb75716sup17.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016004692/hb75717sup18.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016004692/hb75718sup19.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016004692/hb75719sup20.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016004692/hb757110sup21.cmlSupporting information file. DOI: 10.1107/S2056989016004692/hb7571sup22.pdfSupporting information file. DOI: 1425668, 1425667, 1425666, 1425665, 1425664, 1425663, 1425662, 1425661, 1425660, 1425659CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Infected T cells, both healthy and dead or dying, were taken up through viral envelope glycoprotein-receptor-independent interactions, implying a mechanism distinct from conventional virological synapse formation. Macrophages infected by this cell-to-cell route were highly permissive for both CCR5-using macrophage-tropic and otherwise weakly macrophage-tropic transmitted/founder viruses but restrictive for nonmacrophage-tropic CXCR4-using virus. These results have implications for establishment of the macrophage reservoir and HIV-1 dissemination in vivo.Macrophages contribute to HIV-1 pathogenesis by forming a viral reservoir and mediating neurological disorders. Cell-free HIV-1 infection of macrophages is inefficient, in part due to low plasma membrane expression of viral entry receptors. We find that macrophages selectively capture and engulf HIV-1-infected CD4  •Macrophages selectively capture and engulf HIV-1-infected T cells•Uptake of HIV-1-infected T cells drives efficient macrophage infection•T cell capture is viral Env independent; macrophage infection is Env-receptor dependent•This represents a route for macrophage infection by transmitted/founder viruses Tissue macrophages play an important role in HIV-1 infection as viral reservoirs. Baxter et al find that engulfment of HIV-1-infected T cells represents an efficient route by which macrophages may become infected. This mode of spread enables weakly macrophage-tropic transmitted/founder viruses, implicated in viral transmission, to infect macrophages. Macrophages are scavengers that phagocytose dead and dying cells during normal tissue homeostasis, and detect and eliminate infected cells in their role as innate immune sentinels . In immu+ T cells that is more efficient than cell-free spread is cell-to-cell spread  or R5 T/F virus (HIV-1CH077mCherry+) and live-cell imaged over 2 hr. NL4.3/GFP+ Jurkats. Similarly, an MDM engulfs two HIV-1CH077/mCherry+ Jurkats  R5 HIV-1BaL, and processed for imaging as described in −) healthy (Caspase3−/PS−/LD−) T cells were selectively ignored by MDMs compared to either infected/healthy (p < 0.01) or uninfected/dead/dying (p < 0.05) captured T cells . Strikingly, infected/dead/dying T cells were highly significantly captured by MDMs compared to uninfected/healthy (∼50-fold over p < 0.001) and at a significantly higher frequency than either infected/healthy T cells (p < 0.01) or uninfected/dead/dying cells (p < 0.05)  or primary CD4 Jurkats  or an HI+ T cell . These rchanisms  and macrchanisms . We test < 0.05) E. These + T cell capture by MDM, we quantified T cell uptake using qPCR of MDM-associated viral (v)DNA DNA A or luci was released from MDM directly exposed to infected T cells than exposed to cell-free virus -tropic and nonmacrophage (NM)-tropic HIV-1-infected T cells associated with MDM. We confirmed that MDM cocultured with M-tropic HIV-1BaL+ T cells for 6 days were productively infected: ∼25% of MDMs expressed cytoplasmic Gag and released free Gag p24, and these signals were eliminated by AZT treatment  not associated with a CD3 signal , thereby excluding CD3+ debris  were Gag+  and infected with the following viruses: WT HIV-1BaL or HIV-1IIIB , 5–50 ng CA-p24 per 1 × 107 CD4+ T cells for 7 days in complete RPMI 1640/10% fetal calf serum/10 U/ml IL-2 (CFAR). Replication-competent IMC stably expressing Renilla reniformis luciferase in an isogenic NL4.3 backbone (NL-LucR.T2A) encoding diverse env ectodomains in cis . For live-cell imaging experiments, Jurkat-Tat-R5 or primary CD4+ T cells were infected with the X4 HIV-1NL4.3-GFP or mCherry IMC expressing R5 T/F Env CH077 (NL4.3-RFP (HIV-1CH077/mCherry) by magnetofection  and 50 μl lysate mixed with 50 μl Ren-Glo luciferase solution (Promega) at RT and activity  measured after 10 min. For GFP-reporting of MDM infection, PHA/IL-2-activated CD4+ T cells were synchronously magnetofected or mock infected for 48 hr with HIV-1BaL-GFP. T cells were washed, added to autologous MDM 1:1, and cocultured for 6 hr. Following coculture, free or loosely attached T cells were washed off with 10 mM EDTA, MDMs were cultured, and productive infection was analyzed at day 3 post-coculture by fixing and confocal microscopy. For VSVg-mediated HIV-1 transduction, PHA/IL-2-activated CD4+ T cells were synchronously magnetofected for 48 hr with HIV-1BaL or VSV-G pseudotyped HIV-1BaLΔEnv  or mock infected. Infected CD4+ T cells were added to autologous MDM (1:1) and cocultured for 3 hr. MDMs were washed before lifting in 5 mM EDTA/12 mM lidocaine, fixation, permeabilization, staining, and analysis by flow cytometry. For p24 ELISA, supernatants were removed, centrifuged, and inactivated with 0.5% empigen/56°C  or cell-free supernatant added directly to target cells. Within-donor variability in T cell infection levels between IMCs in MDM infection readouts was normalized in  primers , and resgen/56°C .Inhibitors of MDM uptake were jasplakinolide  and 5-N-ethyl-N-isopropyl amiloride . Inhibitors of HIV-1 Env-receptor interactions were 13B.8.2, CD4-specific gp120 blocking mAb (10 μg/ml Beckman-Coulter); soluble CD4 ; Tak-779 ; 2G12 ; and T20 . Relevant isotype controls were used at matched concentrations. MDM Fc receptors were blocked in 10 μg/ml pooled human IgG at 37°C for 1 hr. Inhibitors were preincubated with MDMs and/or T cells at 37°C and maintained during coculture (HIV-1 Env receptor inhibitors) or washed out after preincubation with MDMs (MDM uptake inhibitors). MDMs were pretreated with 13B8.2, T20, Tak-779, EIPA, or jasplakinolide. HIV-1-infected T cells were pretreated with sCD4, 2G12, or T20.4Cl in PBS for 20 min, nuclei stained with 1 μg/ml Hoechst in PBS, and cells permeabilized in wash buffer . Samples were stained for CD3 , mouse anti-HIV-1 Gag p17  with anti-mouse Alexa 488/647-IgG1 or -IgG2a (Invitrogen). Alternatively, directly conjugated mouse anti-HIV-1 Gag KC57-FITC (Beckman-Coulter) and phalloidin-TRITC (Sigma) were used. Coverslips were mounted in ProLongGold Antifade (Invitrogen) and analyzed using an Olympus FV1000. Images were acquired with a 60× oil-immersion objective and processed in Olympus Fluoview v2.0b.Performed as described previously in BaL-infected CD4+ T cells were unlabeled or labeled with anti-PS-FITC (Millipore), Live/Dead (LD)-Red (Invitrogen). In PS prelabel experiments, cells were postlabeled with CD3-PerCP Cy5.5 (BD Biosciences), active Caspase3-APC (BD Biosciences), and Gag  KC57-PE (Beckman Coulter). For LD experiments or experiments with no T cell prelabel, cells were labeled with CD3-APC (BD Biosciences), Caspase3-PE (BD Biosciences), and Gag KC57-FITC (Beckman Coulter). MDMs and T cells were cocultured (1:1) for 3 hr, washed extensively with PBS/EDTA (10 mM), and lifted with cold PBS/EDTA (5 mM)/lidocaine (12 mM). Cells were fixed in 4% PFA and permeabilized. T cell phenotypes  or 6 hr HIV-1BaL+ T cell supernatants were cocultured with MDM for 6 hr prior to extensive washing. MDMs were maintained for 3 to 6 days, lifted, fixed (as above), and stained with CD3-APC and Gag KC57-FITC. Samples were processed using ImageStream 100 or ImageStreamX and analyzed with IDEAS 4.0 or 5.0 software (Amnis).MDM were differentiated for 7 to 10 days. Autologous HIV-1enotypes A–S1H andenotypes I–S1N werBaL+ CD4+ T cells were labeled with PS-FITC, LD-Red, or unstained prior to coculture and prepared as for ImageStream. Antibodies used were as follows: CD14-APC (BioLegend), CD14-FITC (BD Biosciences), CD3-FITC, CD3-APC, KC57-FITC/PE, CD4-APC, or CCR5-FITC, together with appropriate fluorochrome-conjugated isotype controls. Cells were gated according to scatter and staining for CD14 (MDM) or CD3 (T cell). Samples were collected on a FACSCalibur (BD) and analyzed with FlowJo.Autologous HIV-1All analyses were performed with Graphpad Prism V5 or V6, and data were initially interrogated for normal distribution. For group-wise comparisons, one-way ANOVA with Bonferonni post hoc test (parametric), Kruskal-Wallis one-way ANOVA with Dunn’s post hoc test (nonparametric), or two-way ANOVA with Bonferonni post hoc test (parametric) were used to account for multiple comparisons. Pairwise comparisons were done with Student’s t test or Mann-Whitney U. When comparing to a hypothetical mean, one-sampled t tests with Sidak’s post hoc test correction were used. All tests were two-tailed with alpha < 0.05 considered significant.Q.J.S. and F.G. conceived the study; Q.J.S., F.G., A.E.B., R.A.R., and C.J.A.D. conceived, designed, and carried out experimental approaches; and Q.J.S., A.E.B., R.A.R., and C.J.A.D. wrote the paper. F.G., A.E.B., R.A.R., C.J.A.D., M.D.M., and C.W. performed, analyzed, and interpreted the experiments, and C.O., J.C.K., J.L.P., A.F., and D.E.K. supplied novel reagents. All authors have read, edited, and approved the final manuscript.The authors declare no competing financial interests.


Centrosymmetric dimers with a central  7H5BrO3, into centrosymmetric dimers which display a central R22(8) ring motif. In addition, there is an intramolecular hydroxyl–carboxyl O—H⋯O interaction present. A comparison with the crystal structures of 59 other substituted derivatives of salicylic acid shows that both the centrosymmetric carboxyl–carboxyl O—H⋯O dimer and the stacking mode of molecules along the short a axis observed in the title structure are frequent packing motifs in this set.Mutual carboxyl–carboxyl O—H⋯O hydrogen bonds link the molecules of the title compound, C Members of this class have served as model compounds for studies of crystal polymorphism  connection involving a pair of antiparallel interactions between their carboxyl groups  which involves the hydroxyl group at C2. The sheets are stacked in a parallel fashion in the a-axis direction with an intersheet separation of 3.798 (4) Å which corresponds to the length of this axis.Neighbouring molecules are linked to one another by a two-point O2—H2⋯O3 Fig. 3a which nR at a ring position n  is encoded nn-R . The title compound of the present study is denoted 3-Br in this system. Our own survey of the Cambridge Structural Database  which is present in all compounds of the set. A quantitative descriptor, the dissimilarity index x10 O—H⋯O(carboxyl) hydrogen-bonded dimers with a central et al., 2001x10 values are smaller than 12 (Table S1 of the Supporting information). In keeping with the nomenclature introduced by Montis & Hursthouse ]. We have identified another 21 examples of the same stacking mode (Table S1 of the Supporting Information) and the 13 best matches with x10 > 12 are listed in Table 2right)].A one-periodic SC, denoted X11 by Montis & Hursthouse 2012, describet al., 2008et al., 2005a. The corresponding x10 values of 11.8 and 12.4 for this 2-periodic SC [denoted S2 in Fig. 5left)] indicate a relaxed form of geometrical similarity, which is consistent with the accommodation of additional halogen substituents in the planes of 3,5-Cl and 3,5-Br. Moreover, the short C4—H4⋯O1 contact found in 3-Br (see above) is replaced by other close contacts in the S2 instances of 3,5-Br and 3,5-Cl. Table S2 of the Supporting information contains the corresponding crystallographic parameters associated with SC S2. A graphical overview of the packing relationships involving 3-Br and their interdependencies is given in Fig. 5left).Other noteworthy packing relationships exist between 3-Br and the structures of 3,5-Br : 6.87 , 5.3 , 7.80 , 11.5  p.p.m. 13C NMR : 110.1, 114.3, 120.2 (CH), 129.7 (CH), 138.5 (CH), 157.6, 171.6 p.p.m. 1H NMR : 6.85 , 7.79 , 7.92 , 11.07  p.p.m. 13C NMR : 111.8, 112.8, 120.7 (CH), 130.6 (CH), 140.5 (CH), 159.0, 174.1 p.p.m. IR (neat): 2855, 2526, 1653, 1603, 1428, 1298, 1243, 1153, 851, 744, 677, 469 cm−1.Uiso(H) = 1.2 Ueq(C). The H atoms attached to O were identified from difference Fourier maps and their positions refined with restrained distances [O—H 0.86 (2) Å] and their isotropic thermal displacement parameters were refined freely.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015007331/wm5143sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989015007331/wm5143Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015007331/wm5143Isup3.pdfSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015007331/wm5143Isup4.cmlSupporting information file. DOI: 1059331CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Class II phosphoinositide 3-kinase α-isoform (PI3K-C2α) is involved in regulating KCl-induced vascular smooth muscle contraction. The current study was to investigate the effects of sevoflurane (SEVO) and isoflurane (ISO) on KCl-elicited PI3KC2α mediated vasoconstriction in rat aortic smooth muscle.Isometric force, in the absence or presence of SEVO or ISO , PI3K inhibitor LY294002, Rho kinase inhibitor Y27632, and membrane translocation of PI3K-p85, PI3K-C2α, Rho kinase (Rock II), or phosphorylation of MYPT1/Thr853, MYPT1/Thr696, CPI-17/Thr38 and MLC in response to KCl (60 mM) was measured by using isometric force transducer and western blotting analysis, respectively.p <0.05, p < 0.01, respectively). Not only Y27632 (1 uM), but also LY294002 (1 mM), inhibited KCl-induced Rock-II membrane translocation (p < 0.01). SEVO and ISO inhibited KCl-stimulated MLC phosphorylation, PI3K-C2α and Rock-II,not PI3K p85 membrane translocation in a concentration-dependent manner in rat aorta. Both SEVO and ISO suppressed the MYPT1/Thr853, not MYPT1/Thr696 and CPI-17/Thr38, MLC phosphorylation in response to KCl.KCl elicited a rapid and sustained contraction of rat aortic smooth muscle that was inhibited by both SEVO and ISO in a concentration-dependent manner, and also suppressed by LY294002 (1 mM) and Y27632 (1 uM). LY294002 (1 mM) and Y27632 (1 uM) also inhibited KCl-induced MLC phosphorylation. LY294002 (1 mM) inhibited KCl-induced PI3K-p85, PI3K-C2α membrane translocation in response to KCl (PI3K-C2α mediates part of SEVO and ISO-mediated vasodilation in rat aorta. The cellular mechanisms underlying the inhibitory effect of volatile anesthetics might be mediated by KCl/PI3K-C2α/Rho kinase/MYPT1/MLC pathway. Membrane depolarization by KCl increases Ca2+ influx via voltage-dependent Ca2+ channel receptors on VSM cells, leading to an increase in cytosolic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i activates Ca2+-calmodulin-dependent myosin light chain kinase, resulting in MLC phosphorylation and vascular contraction i in VSM [2+-induced Ca2+ sensitization in regulating VSM contraction. The current study involved investigating the effects of volatile anesthetic modulation of PI3K subunit and Rho kinase activity on vasoconstriction in response to KCl in rat isolated aortic smooth muscle.We previously demonstrated that SEVO inhibited GTPγ S-stimulated Rho-Rho kinase-mediated vasoconstriction in isolated rat aortic VSM . We alsoi in VSM . We recei in VSM . HoweverThis protocol was approved by Shandong University Animal Care and Use Committee and Qilu Hospital of Shandong University Medical Ethics Committee (No. KYLL-2013-51). Aortic rings and strips were prepared according to previous protocols –18. Male2, 2.5; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 24.8; and dextrose 10) at 37 °C and gassed with a mixture of 95 % (v/v) O2 and 5 % (v/v) CO2 with a fresh gas flow of 2 L/min. After 60 min equilibration, with the bathing fluid replaced every 20 min, the aortic rings were incubated with KCl (60 mM) to access their overall contractile responsiveness. Removal of the endothelium was confirmed with 3 × 10−7 M phenylephrine pre-contracted vessels that showed a lack of relaxation in the presence of 10−5 M acetylcholine. To examine the effect of anesthetics on KCl-induced contraction, six aortic rings from each individual rat (n = 7) were randomly exposed to 0, 1, 2, and 3 minimum alveolar concentration (MAC) of each anesthetic, 1 mM LY294002 (PI3K inhibitor), or 1 μM Y27632 (Rho kinase inhibitor) for 15 min before the addition of KCl  was used as an agonist according to previous results . KCl-elin = 8–12) and 0.19, 0.39, and 0.56 mM for gas concentrations of 1.2 % (1 MAC), 2.3 % (2 MAC), and 3.5 % (3 MAC) ISO, respectively (n = 8–12), after 15 min of equilibration.SEVO or ISO was delivered into the gas mixture with a fresh gas flow of 2 L/min via a calibrated agent-specific vaporizer  to aerate the KBS with equivalent human MAC. The concentration of the resulting gas mixture was monitored and adjusted using an Atom 303 anesthetic agent monitor . Our previous data using the same experimental system indicated that the concentrations of SEVO or ISO in KBS measured by gas chromatography  were 0.17, 0.28, and 0.41 mM for gas concentrations of 1.7 % (1 MAC), 3.4 % (2 MAC), and 5.1 % (3 MAC) SEVO, respectively  were bathed in oxygenated KBS and were equilibrated for 60 min before the start of the experiment. One strip was obtained from each animal.Previous data demonstrated that KCl (60 mM) induced a rapid increase in MLC phosphorylation and reached peak levels approximately 5 min after exposure to KCl . TherefoIn each experiment, proteins (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis 12 % or 5 %) and transferred to nitrocellulose membranes. Membranes were incubated with anti-MLC, anti-CPI-17, anti-MYPT1/Thr696, anti-MYPT1/Thr853 , and antiphospho-MLC, antiphospho-CPI-17, antiphospho-MYPT1/Thr696, and antiphospho-MYPT1/Thr853 antibodies  for 2 h, followed by incubation with a horseradish peroxidase-conjugated antibody (1:2000) for 1 h [ % or 5 %The densities of immunoreactive bands were detected using chemiluminescence  and were assessed with image analysis software . Bands with molecular weights of 17 kDa, 20 kDa, and 110 kDa were identified as CPI-17, MLC, and MYPT1, respectively. The ratios of phosphorylated to total CPI-17, MLC, and MYPT1 were used as an indicator of activation of each enzyme and expressed as the percentage relative to the baseline control level (referred to as 100 %).For enzyme membrane translocation, PI3K and Rho kinase (Rock II) activity were measured using western blotting analysis as described previously . Briefly2, 100 mM NaCl, 2 mM EDTA, 1 mM 4-(2-aminoethyl) benzonesulfonyl fluoride, 20 μg/mL leupeptin, 20 μg/mL aprotinin, and 1 mM Na3VO4. Homogenates were centrifuged at 13,000 g for 15 min at 4 °C, and the supernatants were collected and centrifuged at 100,000 g for 30 min at 4 °C again. The supernatant (cytosolic fraction) was removed, and the pellet (membrane fraction) was re-suspended using the same lysis buffer. The protein concentrations of each fraction were determined using the bicinchoninic acid method [Frozen aortas were cut into small pieces and were homogenized in ice-cold lysis buffer containing 1 mM Tris/HCl, pH 7.5, 5.5 mM MgCld method .Equal amounts of total protein (30 μg) were used for every sample in each experiment. Proteins were separated by 7.5 % or 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to a nitrocellulose membrane. The membrane was treated with anti-PI3K-p85α, anti-PI3K-C2α, and anti-Rock-II antibodies  for 4 h, followed by incubation with a horseradish peroxidase-conjugated antibody (1:2000) for 1 h. Immunoreactive bands were detected using chemiluminescence  and were assessed with image analysis software . The amount of PI3K-p85α, PI3K-C2α, and Rock-II on the membrane was expressed as a percentage of the total value .SEVO and ISO were purchased from Dainabot Company Limited . Polyclonal antibodies against phospho-MLC (Thr18/Ser19), phospho-MYPT1 (Thr853), phospho-MYPT1 (Thr696), phospho-CPI-17 (Thr38), MLC2 (FL-172), MYPT1 (H-130), CPI-17 (H60), PI3K-p85α, and Rock-II, and the secondary antibody labeled with horseradish peroxidase (HRP) were all obtained from Santa Cruz Biotechnology, Inc. . PI3K-C2α was purchased from Abcam, Inc. . LY294002 was provided from Merck-Calbiochem Biosciences . Y27632 was purchased from Calbiochem-Novabiochem Corporation . All other reagents for experiments and western blotting were of analytical grade.n) refers to the number of rats from which the aortas were harvested. Data are presented as the mean ± standard deviation and were analyzed using the Dunnett test after analysis of variance for multiple comparison of isometric tension or protein phosphorylation. A p-value < 0.05 was considered significant.Data analyses were performed using the software program StatMate . The sample size    inhibited KCl-induced PI3K-p85 and PI3K-C2α membrane translocation in response to KCl (01) Fig. .2+-induced Ca2+ sensitization induced by volatile anesthetics contributes to its vasodilatory effect.The current study demonstrated that both SEVO and ISO inhibit KCl-induced MLC phosphorylation and vasoconstriction by modulating PI3K-C2α and Rho kinase activity in the rat aorta. These findings suggest that the inhibition of Ca2+ sensitivity. Because specific PI3K-C2α-inhibitors do not exist, it remains unclear whether the reduced MLC phosphorylation and vasoconstriction depends on class II PI3K-C2α-inhibition or inhibition of class I PI3K.Several studies demonstrate that LY294002 and Y27632 inhibit KCl-induced vasoconstriction by suppressing MYPT1 and CPI-17 phosphorylation in rat or rabbit aortic smooth muscle , 11. LY22+-induced Ca2+ sensitization [2+/PI3K-C2α/Rho kinase/MYPT1/Thr853 pathway.Accumulating evidence demonstrates that PI3K-C2α and Rho kinase, or PKC, MYPT1/Thr853, and CPI-17 are involved in vasoconstriction by regulation of Catization , 23–25. tization , 27. Taktization . It is p2+ sensitivity by regulating Rho kinase and PKC activity [2+ changes in the diabetic rat aorta [2+ sensitization, not Ca2+-dependent mechanisms, is involved in modulating vascular tension by SEV.Several reports also showed that SEVO decreased myofilament Caactivity , 17, 18.at aorta . These d2+ sensitivity mechanisms is mainly mediated by increasing Ca2+ influx via voltage-dependent calcium channels [2+-dependent pathway and not through the Ca2+ sensitivity pathway. In contrast to the above results, we recently demonstrated that ISO inhibits Ang II-induced vasoconstriction through regulating the phosphorylation of the MLCP subunit MYPT1/Thr853 [2+-induced Ca2+ sensitization pathway was also involved in the regulation of vasoconstriction by ISO.Evidence also demonstrates that ISO inhibition of agonist-induced contraction of Cachannels , 30. We channels . These d1/Thr853 . Further2+-induced Ca2+ sensitization through modulating PI3K-C2α, Rho kinase activity, and MYPT1/Thr853 phosphorylation in rat vascular smooth muscle.Overall, our data, in combination with other investigations, reveal that inhibition of KCl-elicited vasoconstriction by volatile anesthetics may involve the modulation of PI3K-C2α and/or Rho-Rho kinase activity, the upstream activators of MLCP , 11, 25.in vivo conditions. However, we used endothelium-denuded samples to avoid the effects of the endothelium. (2) Moreover, pre-arteriolar resistance arteries, not conduit arteries, mainly determine peripheral vascular resistance. Therefore, the current results obtained from endothelium-denuded aortas cannot be used to directly extrapolate to the in vivo condition. (3) Furthermore, the aortic rings are not necessarily reflective of the physiology of resistance arteries and thus may not be ideally suited to address issues related to the clinical hemodynamic effects of volatile anesthetics.However, this study has some limitations: (1) the aim of this study was to elucidate the cellular mechanisms responsible for anesthetic-induced inhibition of vasoconstriction. Therefore, we used endothelium-denuded aortic strips or rings for this study. Indeed, the endothelium plays an important role in regulating systemic vascular tone under In conclusion, our study demonstrates that both SEVO and ISO inhibit KCl-induced PI3K-C2α-participation, Rho kinase-mediated MLC phosphorylation, and vasoconstriction in rat aortic smooth muscle. The intracellular mechanisms of regulating VSM contraction of volatile anesthetics may be mediated by the KCl/PI3K-C2α/Rho kinase/MLCP/MLC pathway.Ang II, Angiotensin II; BCA, bicinchoninic acid; CaM, Ca2 + -calmodulin; CPI-17/Thr38, PKC-potentiated inhibitory protein/Thr38; GPCR, G protein-coupled receptors; ISO, isoflurane; MAC, minimum alveolar concentration; MLC, myosin light chain; MLCK, myosin light chain kinase; MLCP, MLC phosphatase; MYPT1/Thr696, myosin phosphatase target subunit/Thr696; MYPT1/Thr853, myosin phosphatase target subunit/Thr853; PI3K-C2α, Class II phosphoinositide 3-kinase α-isoform; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEVO, sevoflurane; VSM, vascular smooth muscle