The performance of H2O2 production remains limited by the inertness of graphitic carbon nitride (CN) towards the adsorption and activation of O2. In this work, a photocatalyst comprising of β-cyclodextrin (β-CD)-modified CN with supporting Au co-catalyst (Au/β-CD-CN) has been utilized for effective H2O2 production under visible light irradiation. The static contact angle measurement suggested that β-CD modification increased the hydrophobicity of the CN photocatalyst as well as its affinity to oxygen gas, leading to an increase in H2O2 production. The rate of H2O2 production reached more than 0.1 mM/h under visible-light irradiation. The electron spin resonance spectra indicated that H2O2 was directly formed via a 2-electron oxygen reduction reaction (ORR) over the Au/β-CD-CN photocatalyst.Photocatalytic hydrogen peroxide (H T. T22]. T−1 can be correlated to the vibrational absorptions of tri-s-triazine subunits. The typical absorption peaks of CN at 1570 cm−1 and 1635 cm−1 correspond to C–N and C=N, respectively. The vibration bands at 1236 cm−1, 1316 cm−1, and 1403 cm−1 belong to the aromatic C–N heterocycles. The formation of a broad peak at 3173 cm−1 is linked to N–H stretching. All the above peaks arise in the β-CD-CN. After CN modification by β-CD, new adsorption bands at around 550 cm−1, 1027 cm−1, and 1157 cm−1 are related to the β-phase formation, C–O–C, and C–C/C–O. Those are attributed to a large number of hydroxyl groups of β-CD [−1. The broad vibration band at 3392 cm−1 belongs to O–H stretching vibration, which also arises in the spectra of the pure β-CD sample. These results clearly indicate the formation of β-CD-CN [ of β-CD ,29. Due  β-CD-CN .The optical absorption of CN, β-CD-CN, and 0.05% Au/β-CD-CN were measured by UV–vis. All samples show wide absorption in the visible region a. The ab2O2 production [The photoluminescence (PL) spectra of CN, β-CD-CN, and 0.05% Au/β-CD-CN nanocomposites are presented in oduction .2O2 production [A water contact angle test was carried out to study the change in the Au/β-CD-CN surface property. As shown in oduction .2O2 production over M/β-CD-CN composite was tested under visible light and a spectrophotometric method was used to quantify the H2O2 concentrations [2O2 and absorbance exhibited a good linear relationship with R-square of 0.9988  showed the best photocatalytic activity for H2O2 production and the reaction rate reached more than 0.1 mM/h. Moreover, the amount of Au loading on β-CD-CN is critical to the photocatalytic activity  and holes (h+) are separated (Equation (1)), e− is positioned at the C1 and N4 positions of the triazine ring, and h+ is positioned at the N2 and N6 positions, that is, an electron (e−)-hole (h+) pair is formed. However, electrons easily recombine with holes. After CN is loaded with Au, electrons (e−) can be rapidly transferred from C1 and N4 to Au, thereby effectively inhibiting the recombination of electrons and holes. Holes (h+) capture α-H and β-H in CH3CH2OH, and CH3CH2OH generates CH3CHO (Equation (2)). Immediately after β-CD modification, a hydrogen bond formed between the amino group of CN and the hydroxyl group of β-CD. The β-CD-CN plays an excellent adsorption role with oxygen. Electrons (e−) reduce O2 to produce H2O2 and complete the photocatalytic cycle (Equation (3)).The mechanism of Au/β-CD-CN photocatalytic activity of H2O2 production under visible light. The β-CD modified CN showed a hydrophobic property, which was beneficial for adsorption with oxygen to improve H2O2 production. Significantly, the optimal photocatalyst with the Au loading amount as low as 0.05% reached the maximal H2O2 production rate. Simultaneously, Au/β-CD-CN exhibited an inertness decomposition rate of H2O2 under visible light. The ESR test demonstrated that H2O2 was directly formed via 2-electron oxygen reduction over photocatalysis. This article provides a new method that loading Au on β-CD-modified CN can increase the adsorption and activation of O2 to improve photocatalytic activity for H2O2 production.Au/β-CD-CN was successfully obtained for improving photocatalytic activity for H


Fifteen 4-(2-methoxyphenyl)piperazin-1-ium salts with organic anions exhibit a range of hydrogen-bonded supramolecular assemblies in the form of finite aggregates, a chain of rings, ribbons, sheets and three-dimensional networks. 11H17N2O+·C7H4ClO2− (I) and C11H17N2O+·C7H4BrO2− (II), and the 4-iodobenzoate salt C11H17N2O+·C7H4IO2− (III), the ions are linked by N—H⋯O hydrogen bonds, forming centrosymmetric R44(12) four-ion aggregates; a similar aggregate is formed in the 2-chlorobenzoate salt (V), isomeric with (I). In the 2-fluorobenzoate salt C11H17N2O+·C7H4FO2− (IV), and the isomorphous pair of salts, the 2-bromobenzoate (VI), isomeric with (II) and 2-iodobenzoate (VII), isomeric with (III), N—H⋯O and C—H⋯π(arene) interactions link the components into three-dimensional arrays. Four-ion R44(12) aggregates are also found in the 2-methylbenzoate, 4-aminobenzoate and 4-nitrobenzoate salts, C11H17N2O+·C8H7O2− (VIII), C11H17N2O+·C7H6NO2− (IX) and C11H17N2O+·C7H4NO4− (X), but those in (IX) are linked into complex sheets by an additional N—H⋯O hydrogen bond. In the 3,5-dinitrobenzoate salt, C11H17N2O+·C7H3N2O6−·2H2O (XI), N—H⋯O and O—H⋯O hydrogen bonds link the components into a complex ribbon structure. In the picrate salt, C11H17N2O+·C6H2N3O7− (XII), the four-ion aggregates are linked into chains of rings by C—H⋯O hydrogen bonds. In the hydrogen maleate salt, C11H17N2O+·C4H3O4− (XIII), two- and three-centre hydrogen bonds link the ions into a ribbon structure while both anions contain very short but asymmetric O—H⋯O hydrogen bonds, having O⋯O distances of 2.4447 (16) and 2.4707 (17) Å. O—H⋯O Hydrogen bonds link the anions in the hydrogen fumarate salt (XIV), isomeric with (XIII), into chains that are linked into sheets via N—H⋯O hydrogen bonds. In the hydrogen -tartrate salt, C11H17N2O+·C4H5O6−·1.698H2O (XV), the anions are linked into sheets by O—H⋯O hydrogen bonds. Comparisons are made with the structures of some related compounds.Fifteen 4-(2-methoxyphenyl)piperazin-1-ium salts containing organic anions have been prepared and structurally characterized. In the isostructural 4-chlorobenzoate and 4-bromobenzoate salts, C N-(4-methoxyphenyl)piperazine (4-MeOPP) piperazine (4-FPP) piperazine (2-MeOPP), which has been used as a building block in the synthesis of both 5-HT1A receptor ligands PLATON Z′ = 2 . In sharp contrast to compound (XIII)R,3R)-tartaric acid crystallizes as a dihydrate 2 Fig. 13. A searce Fig. 15.N-aryl substituent in an equatorial site. In the 2-methoxyphenyl units, the methoxy C atom is always close to coplanar with the adjacent aryl ring: the displacement of this atom from the plane of the ring ranges from 0.038 (5) Å in compound (I)ca 10°, as is usually observed in planar or near-planar alkoxyarenes The two independent ions in compound (XIII)D—H⋯A angles that are significantly less than 140°, as the interaction energies associated with such contacts are likely to be very low, so that these cannot be regarded as structurally significant The supramolecular assembly in the salts (I)–(XV) is based on N—H⋯O and O—H⋯O hydrogen bonds augmented in a number of cases by C—H⋯O and C—H⋯π(arene) hydrogen bonds. In general, we have discounted hydrogen bonds having et al., 1990et al., 1995D—H⋯A angle and is thus not structurally significant et al., 1998abet al., 2000The hydrogen bonding involving the two disorder components in compound (IV)n Fig. 18, and then Fig. 18. The N—Hn Fig. 18, and the1 screw axis along  to form a 1 screw axis along  are linked by a C—H⋯O hydrogen bond to form a The ion pairs in compounds (VI)] Fig. 21. In addi] Fig. 22, while t] Fig. 22. The comThe ions in compound (VIII)s Fig. 26. AggregaZ′ = 2, each of the anions contains a very short O—H⋯O hydrogen bond, although in each of these interactions the two O—H distances are significantly different  alternate with n, 1/2), where n represents an integer in each case  and the disordered anions are similarly linked into a chain along . The two types of chain, which alternate along the [010] direction, are linked by the cations to form a sheet of z < 1.0 and having a hydrogen-bonded layer in the centre with the aryl groups on the outside surfaces: there are no direction-specific interactions between adjacent sheets.In compound (XIII)t Table 1. The supe Fig. 27. Analysi) Fig. 28. In the d Fig. 29. The catIn summary, therefore, the hydrogen-bonded assembly is finite, or zero-dimensional in compounds (I)–(III), (V)N-(4-methoxyphenyl)piperazine (4-MeOPP) and the analogous N-(4-fluorophenyl)piperazine (4-FPP). The salts formed between 4-MeOPP and the benzoic acids 4-XC6H4COOH, where X = H, F, Cl, and Br, all crystallize as stoichiometric monohydrates and they are all isomorphous in space group Pet al., 2019et al., 2020et al., 2020et al., 2020Z′ = 1 Z′ = 1: a combination of N—H⋯O and O—H⋯O and C—H⋯π(arene) hydrogen bonds links the ions into a three-dimensional structure, as opposed to the two-dimensional structure of (XIV)et al., 2020et al., 2020et al., 2020R,3R)-tartaric acid and 4-FPP crystallizes as a monohydrate piperazine  in methanol (10 ml) were mixed with an equimolar quantity of the appropriate acid  also dissolved in methanol (10 ml). These mixtures were then heated briefly at 323 K with magnetic stirring and then set aside to crystallize at room temperature. The resulting products were then collected by filtration and dried in air. Crystals suitable for single-crystal X-ray diffraction were grown by slow evaporation, at ambient temperature and in the presence of air, of solutions in acetone/acetonitrile  for (I)v/v) for (II)v/v) for (III)v/v) for (IV)v/v) for (VI)v/v) for (IX)v/v) for (XI)All reagents were obtained commercially, and all were used as received. For the synthesis of compounds (I)–(XV), solutions of x parameter  − (I−)]/[(I+) + (I−)] x = 0.004 (5) calculated using 919 coefficients, and for (VII)x = 0.004 (10) calculated using 1045 coefficients. For the minor disorder component in compound (IV)3), 0.97 Å (CH2) or 0.98 Å , and with Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups, which were permitted to rotate but not to tilt, and 1.2 for all other H atoms bonded to C atoms: the H atoms in the minor disorder component of compound (IV)UisoH = 1.2Ueq(N), but in all other compounds, the atomic coordinates of the H atoms bonded to N atoms were refined with UisoH = 1.2Ueq(N), giving the N—H distances shown in Table 1Uiso(H) = 1.5Ueq(O), giving the O—H distances shown in Table 1Uiso(H) = 1.5Ueq(O).Crystal data, data collection and refinement details are summarized in Table 210.1107/S2056989020014097/hb7950sup1.cifCrystal structure: contains datablock(s) global, I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV. DOI: 10.1107/S2056989020014097/hb7950Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020014097/hb7950IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989020014097/hb7950IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989020014097/hb7950IVsup5.hklStructure factors: contains datablock(s) IV. DOI: 10.1107/S2056989020014097/hb7950Vsup6.hklStructure factors: contains datablock(s) V. DOI: 10.1107/S2056989020014097/hb7950VIsup7.hklStructure factors: contains datablock(s) VI. DOI: 10.1107/S2056989020014097/hb7950VIIsup8.hklStructure factors: contains datablock(s) VII. DOI: 10.1107/S2056989020014097/hb7950VIIIsup9.hklStructure factors: contains datablock(s) VIII. DOI: 10.1107/S2056989020014097/hb7950IXsup10.hklStructure factors: contains datablock(s) IX. DOI: 10.1107/S2056989020014097/hb7950Xsup11.hklStructure factors: contains datablock(s) X. DOI: 10.1107/S2056989020014097/hb7950XIsup12.hklStructure factors: contains datablock(s) XI. DOI: 10.1107/S2056989020014097/hb7950XIIsup13.hklStructure factors: contains datablock(s) XII. DOI: 10.1107/S2056989020014097/hb7950XIIIsup14.hklStructure factors: contains datablock(s) XIII. DOI: 10.1107/S2056989020014097/hb7950XIVsup15.hklStructure factors: contains datablock(s) XIV. DOI: 10.1107/S2056989020014097/hb7950XVsup16.hklStructure factors: contains datablock(s) XV. DOI: Click here for additional data file.10.1107/S2056989020014097/hb7950Isup17.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014097/hb7950IIsup18.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014097/hb7950IIIsup19.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014097/hb7950IVsup20.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014097/hb7950Vsup21.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014097/hb7950VIsup22.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014097/hb7950VIIsup23.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014097/hb7950VIIIsup24.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014097/hb7950IXsup25.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014097/hb7950Xsup26.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014097/hb7950XIsup27.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014097/hb7950XIIsup28.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014097/hb7950XIIIsup29.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020014097/hb7950XIVsup30.cmlSupporting information file. DOI: 2039887, 2039886, 2039885, 2039884, 2039883, 2039882, 2039881, 2039880, 2039879, 2039878, 2039877, 2039876, 2039875, 2039874, 2039873CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The prepared GA and different acid-doped forms as GA/p(p-PDA) composites were characterized by FT-IR, TGA, and conductivity measurements. The observed FT-IR peaks at 1574 cm−1, and 1491 cm−1, for stretching deformations of quinone and benzene, respectively, confirmed the in situ synthesis of P(p-PDA) polymers within GAs. The conductivity of GAs with 2.17 × 10−4 ± 3.15 × 10−5 S·cm−1 has experienced an approximately 250-fold increase to 5.16 × 10−2 ± 2.72 × 10−3 S·cm−1 after in situ synthesis of p(p-PDA) polymers and with HCl doping. Conductivity values for different types of acid-doped GA/p(p-PDA) composites were compared with the bare p(p-PDA) and their undoped forms. Moreover, the changes in the conductivity of GA and GA/p(p-PDA) composites upon CO2 gas exposure were compared and their sensory potential in terms of response and sensitivity, along with reusability in CO2 detection, were evaluated.In this study, macroporous graphene aerogels (GAs) were synthesized by chemical reduction of graphene oxide sheets and were used as a support material for in situ synthesis of conductive poly(para-phenylenediamine) (p(p-PDA)). The in situ synthesis of p(p-PDA) in GA was carried out by using a simple oxidation polymerization technique. Moreover, the prepared conductive p(p-PDA) polymers in the networks of GAs were doped with various types of acids such as hydrochloric acid (HCl), nitric acid (HNO Preparation of reduced GOs (r-GOs) can be achieved to solve poor conductivity of GOs by treating them with different reducing agents such as hydroquinone, diazene (N2H2), hydrazine(N2H4), sodium borohydride(NaBH4), sodium hydroxide (NaOH), sodium sulfide (Na2S), sodium bisulfate (NaHSO3), ascorbic acid (C6H8O6), and hydroiodic acid (HI) [Over the past decades, carbon-based materials have been the focus of eminent interest in both scientific and industrial fields due to their captivating mechanical, electrical, thermal, and physicochemical properties, as well as their environmentally friendly nature and economically viable accessibility that has collectively led to the emergence of diverse structures including carbon nanotubes (CNTs), carbon dots, mesoporous carbon, and graphene and so on ,2,3,4. Pine form ,6,7. Graine form  includinine form ,10,11, aine form , electroine form ,14, sensine form , biosensine form ,17,18, bine form ,20,21, aine form , e.g., line form ,24, dispine form ,26,27, bine form ,29 and sine form ,31. Howeine form ,32. Chemine form . Severalcid (HI) ,35. GOs cid (HI)  such as cid (HI) , variouscid (HI) , and crycid (HI) .2O4 nanoclusters [2 [3O4 nanoparticles [Aerogels are a specific type of 3D network with ultra-low-density and extreme surface areas, highly porous structure, and great mechanical elasticity, and have been exploited in numerous applications e.g., electronic devices, heat insulation, catalysis, waste management, sensor application, and so on . 3D GO aclusters , TiO2 [4sters [2 , and Co sters [2  nanocryssters [2 , Fe3O4 narticles , and quaarticles , along warticles , poly, sulfuric acid (H2SO4), and phosphoric acid (H3PO4). The prepared GA/p(p-PDA) composites were characterized by Fourier transform infrared (FT-IR) spectroscopy, and thermal gravimetric (TG) analysis. The influences of different acid dopants on the electrical conductivity of GA/p(p-PDA) composites were determined and their sensory potentials against CO2 gas were investigated at ambient pressures and temperatures.In this study, GOs were synthesized from graphite flakes by the modified Hummer method and then used in the fabrication of 3D highly porous GAs upon chemical reduction of the prepared GOs by L-Ascorbic acid (L-AA) as reducing agent and freeze-drying processes. Thereafter, a conductive polymer poly(para-phenylenediamine) (p(p-PDA)) was in situ synthesized within the pores of the prepared GAs followed by doping with several acids such as hydrochloric acid (HCl), nitric acid , sodium hydroxide , potassium permanganate , hydrogen peroxide , and sulfuric acid  were used in the preparation of GOs. L-Ascorbic acid  was used for the reduction of GOs nanosheets in synthesis GAs. P-Phenylenediamine  was used as a monomer for in situ synthesis of p(p-PDA) in the GA networks, and ammonium persulfate  was used as an initiator of oxidative p-PDA polymerization. Hydrochloric acid , nitric acid , sulfuric acid , and phosphoric acid  were used in the doping of in situ synthesized p(p-PDA) within GAs.Graphite flakes , sodium nitrate (NaNOGraphene aerogels (GAs) were prepared by chemical reduction of prepared GOs following the cited literature with some modifications ,67. Conc3, H2SO4, and H3PO4 and added drop by drop in separate p(p-PDA) solutions in an ice bath. Afterward, the prepared p(p-PDA) polymers were collected by centrifugation at 10,000 rpm for 10 min and washed twice with water and acetone, then dried by using a heat gun.The synthesis of conductive poly(p-phenylenediamine) (p(p-PDA)) was carried out in accordance with the literature ,70,71,723, H2SO4, and H3PO4, then stirred for 30 min more at 100 rpm for in situ oxidative polymerization of p-PDA. Various acid-doped GA/p(p-PDA) composites were washed once with water and ethanol then dried in an oven at 50 °C.For in situ synthesis of conductive p(p-PDA) polymers within GA networks, the earlier reports on p(p-PDA) synthesis were followed with some modifications ,70,71,72Morphological properties of GA-based conductive polymer composites were assessed by scanning electron microscopy  operating in a low vacuum mode at a 5 kV accelerating voltage. For the imaging, GA-based samples were mounted on aluminum SEM stubs with the aid of sticky carbon tapes, and the surface of the specimens was coated with gold to a few nanometers of thickness by a sputter coater to prevent excess charge buildup on the surface of specimens, and then samples were analyzed with SEM.−1 with a 4 cm−1 resolution employing attenuated total reflectance of the attached FT-IR spectrometer .Functional groups of GAs and various acid-doped GA/p(p-PDA) composites were ascertained by FT-IR analysis and spectral peaks of these materials were recorded in the range of 650–4000 cmThermal stabilities of GAs and various acid-doped GA/p(p-PDA) composites were determined by a TG analyzer  under continuous purging of nitrogen gas with 200 mL/min flow and the heating rate of 10 °C/min up to 900 °C.Electrical conductivities of GAs, and acid-doped GA/P(p-PDA) composites were measured at room temperature via a computer-controlled electrometer (Keithley 2400 Source-Meter).Conductivity measurements of GAs and various acid-doped GA/p(p-PDA) composites with lengths of 1.24 ± 0.45 cm were performed by attaching small pieces of conductive carbon tape (~3 × 4 mm) to the top and bottom of bare and composite GA samples to connect the electrodes, followed by applying the voltage up to 40 V. The conductivity values were calculated from the slope of the current–voltage (I–V) response curves at the Ohmic (linear) region by using Equations (1) and (2);2 gas was investigated by tracking the change in their conductivity upon CO2 exposure. For this purpose, 0.25 g of bare p(p-PDA) polymers were pelleted under compression of 10 tons of pressure, and the resulting pellet was exposed to CO2 gas for 30 min. The sensory potential of GA/p(p-PDA) composites were also evaluated by 30 min of CO2 gas exposure, which corresponded to 11,980 ppm CO2. The conductivity of p(p-PDA)s, and GA/p(p-PDA) composites, were determined by recording their current–voltage curves via an electrometer and subsequent calculation of conductivity values from Equations (1) and (2). The sensitivity studies of HCl-doped GA/p(p-PDA) composites were conducted by exposing them to CO2 gas for 0.5, 1, 5, 10, 20, 30, 45, and 60 min with the flow rate of 200 mL/min.The potential sensor application of the prepared GA-based composites to CO2 gas for 30 min, and the change in their conductivity was recorded. After that, the used (CO2 exposed) GA/p(p-PDA)-HCl composites were incubated at 60 °C for 2 h for regeneration of their conductivity and recovered conductivities were noted. Following the incubation, the composites were retreated with CO2 gas for another 30 min and the same reuse–regeneration cycles were applied for a total of 5 cycles.Besides, reusability of the GA/p(p-PDA)-HCl composites was also investigated. Briefly, the HCl-doped composite GA samples were treated with CO3, H2SO4, and H3PO4.The schematic illustration describing the preparation of GA/p(p-PDA) composites was given in 2SO4 acid doping as 3.94 ± 0.8 g/g of GA.The amounts of in situ polymerized p(p-PDA) within GA pieces were gravimetrically calculated by the mass difference between dried bare GA and GA/p(p-PDA) composites and the results are reported in 3PO4, HCl, and HNO3 acid solutions, respectively.In the descending order 3.67 ± 0.7 g, 2.08 ± 0.5 g, and 1.78 ± 0.3 g of p(p-PDA) polymers were in situ synthesized by simultaneous doping of H3, GA/p(p-PDA)-H2SO4, and GA/p(p-PDA)-H3PO4 composites are given in Moreover, the SEM analysis of GA-based composites revealed the existence of highly porous structures with pores of varying sizes, in a range of 1–100 µm. The corresponding SEM images of GA, GA/p(p-PDA)-HCl, GA/p(p-PDA)-HNOIt has been reported that high surface area, porosity, and pore volumes of GA-based materials favor diffusion of gases into the porous GA networks ,74. Ther3-, H2SO4-, and H3PO4-doped conductive p(p-PDA) polymers are given in −1 was assigned to symmetric stretching vibrations of =CH, and the peaks at 3032 cm−1 for symmetric and at 3302, and 3201 cm−1 were designated to asymmetric stretching vibrations of N-H bonds. The spectral peaks observed at 1613 cm−1 were attributed to N-H deformation of secondary amines, the peaks at 1502 cm−1 for symmetric stretching vibration of aromatic CC, the peaks at 1327 cm−1 for symmetric stretching of C-N, and the peaks that appeared at 807 cm−1 were assigned to out of plane deformation showing 1,4-distubistution in the benzene ring, respectively [−1, stretching vibrations of benzene at 1503 cm−1, and also the peak at 1303 cm−1 can be assigned to C-N stretching in a secondary aromatic amine, 1098 and 1014 cm−1 for the aromatic C-H in-plane bending modes, and 807 cm−1 for out-of-plane deformations of C-H in the 1,4-disubstituted benzene ring, respectively [The FT-IR spectra of p(p-PDA) and prepared bare HCl-, HNOectively . The FT-ectively . Moreove−1, and the peak for C=O groups from both the carboxylic acid and ketones was observed at 1722 cm−1 [−1 was assigned to the aromatic C=C bonds as a result of skeletal vibrations of unoxidized graphene units. The stretching vibrations at 1042 cm−1 and 873 cm−1, respectively, were attributed to C-O stretching modes of vibrations in the alkoxy and the ether groups of GO [−1 are assigned to O-H groups, and the peak observed at 1758 cm−1 can further stem from the C=O groups, which is noticeable from the FT-IR spectrum of L-AA [−1 and are attributed to the O-H stretching, and the peaks at 1749 cm−1 belong to C=O stretching vibrations. The peaks that appeared at 1616 cm−1 can arise from the skeletal vibration of graphene sheets. From the oxidized graphene, GO sheets to their chemically reduced GA forms, the red shift can be readily noticed for C=O bands with the respective change from 1722 to 1749 cm−1 [−1, stretching deformations of benzene at 1491 cm−1, and C-N stretching modes of a secondary aromatic amine at 1297 cm−1, aromatic C-H in-plane bending modes at 1053 cm−1, and the peaks at 828 cm−1 for out-of-plane deformations of C-H in the 1,4-disubstituted benzene ring can be realized from the given FT-IR spectra [As it is readily seen from the FT-IR spectrum of GO, the O-H stretching vibrations were observed as t broad bands between 3600–3000 cm722 cm−1 ,77. The ps of GO . The FT- of L-AA . For GA,749 cm−1 . Additio spectra . Based uThe TGA thermograms of GA and various acid-doped GA/p(p-PDA) composites were compared in 3-doped GA/p(p-PDA) composites were recorded to show 13.3% weight loss between 110–245 °C, the second and third steps of thermal decomposition occurred between 250–330 °C, and 340–640 °C with 50.1%, and 93.9% cumulative weight losses, respectively. H2SO4-doped GA/p(p-PDA) composites showed approximately 7.5% weight loss up to 185 °C, and heating up to 340 °C resulted in a cumulative weight loss of 77.4%. In addition, in the final decomposition step, more than 99% cumulative weight loss was observed by heating up to 630 °C. For the H3PO4-doped GA/p(p-PDA) composites, 24.3% weight loss was recorded between 130–290 °C. The second step occurred between 540–620 °C with 77.8% cumulative weight loss, and the last step of thermal decomposition was seen between 690–780 °C with a final cumulative weight loss of 91.5% (8.5% remaining residues). Moreover, according to TGA data obtained, H2SO4-doped GA/p(p-PDA) composites were the least thermally stable composite, which was followed by HCl-doped GA/p(p-PDA) composites. Comparatively, the H3PO4- and HNO3-doped GA/p(p-PDA) composites exhibited better thermal stability and higher remaining residues than the ones doped with H2SO4 and HCl, while all of the composites show similar patterns of thermal degradation. The differences seen within thermal degradation profiles of the GA/p(p-PDA) composites can be attributed to the distinctive effects of various acids dopants on the structure and morphology of the in situ synthesized conductive polymers. Particularly, as can be noticeable from the SEM images provided in All of the GA/p(p-PDA) composites shared similar decomposition patterns. The first thermal decomposition of HCl-doped GA/p(p-PDA) composites was recorded between 30–225 °C with 8.4% weight loss, the second step was observed between 230–315 °C with 39.9% cumulative weight loss, and the last step was occurred between 320–630 °C with a final result of more than 99% cumulative weight loss. The HNOCarbon-based graphite, graphene oxide, graphene and/or graphene aerogels have three valence bands where electrons are in high attraction with the central nucleus. There is a free electron called π-electron that does not pass through the Fermi level as in the metals, however, the Fermi level has a narrow band that can easily shift the electron from the valance band to the conduction band and creates electron conductivity . One of 3, p(p-PDA)-H2SO4, and p(p-PDA)-H3PO4 were calculated as 4.46 × 10−8 ± 1.12 × 10−8, 6.46 × 10−9 ± 2.24 × 10−9, 1.99 × 10−8 ± 1.93 × 10−9, and 2.22 × 10−8 ± 1.11 × 10−9 S·cm−1, respectively. From these values, the highest conductivity was measured for the HCl-doped p(p-PDA) polymers, which is approximately 7-fold greater than HNO3, and 2-fold higher than the conductivities of H2SO4-, and H3PO4-doped p(p-PDA) polymers. Electrical conductivity of bare GAs was calculated as 2.17 × 10−4 ± 3.15 × 10−5 S·cm−1, which is much higher than all types of p(p-PDA) polymers with different acid doping. As was expected, conductivity of GAs underwent a significant rise after in situ preparation within GA of various acid-doped forms as GA/p(p-PDA) composites.From these plots, the conductivity values for p(p-PDA) polymers and GA/p(p-PDA) composites the were calculated using Equations (1) and (2), respectively, and compared in 3- H2SO4-, and H3PO4-doped GA/p(p-PDA) composites were calculated to be 5.16 × 10−2 ± 2.72 × 10−3 S·cm−1, 9.19 × 10−4 ± 1.29 × 10−4 S·cm−1, 8.78 × 10−3 ± 1.17 × 10−3 S·cm−1, and 4.11 × 10−4 ± 9.13 × 10−5 S·cm−1, respectively. These values were approximately 250-, 4-, 40-, and 2-fold greater than the conductivity of bare GAs, respectively. As it is in a good agreement with the conductivity of HCl-doped bare p(p-PDA) polymers, the conductivity of in situ prepared HCl-doped GA/p(p-PDA) composites with 5.16 × 10−2 ± 2.72 × 10−3 S·cm−1 is approximately 60-fold, 6-fold, and 125-fold higher than those of HNO3-, H2SO4-, and H3PO4-doped GA/P(p-PDA) composites, respectively. Obviously, different types of acid dopants have imparted distinct conductivity values to the GA based composites. The higher conductivity was observed for in situ prepared HCl-doped GA/p(p-PDA) composites. As mentioned earlier, in the literature, the types of the used acids for doping of conductive polymers have significant effects on size and crystal structure of the conductive polymers, and higher crystallinity was observed in HCl-doped conductive polymers amongst doping of several others [3-doped p(p-PDA) GAs, so is its conductivity. Therewithal, the smaller size of HCl than other used acids can make it a better dopant as it can more readily diffuse into pores of GA composites. Therefore, the highest conductivity values were observed in in situ prepared HCl-doped GA/p(p-PDA) composites.Conductivity of HCl-, HNOl others . As it i2) gas, as one of the most dangerous causatives of global warming, is increasing continuously in the atmosphere. According to the standards reported by the American Association of Heating, Refrigerating and Air Conditioning Engineers (ASHRAE), the concentration of CO2 required for healthy breathing should not exceed 1000 ppm [2 emission is many times higher than this threshold. The CO2 concentration in the atmosphere increased from 414 ppm in May 2019 to 417 ppm in May 2020 [2 increase in the atmosphere. Therefore, the design and use of functional materials that can sense, capture, and store the ways in which the emission of pollutant gases, whose concentration in the atmosphere intensifies daily, endangers the lives of all living creatures. Therefore, CO2 sensors and adsorbent potentials of the prepared bare p(p-PDA) polymers, GAs, and GA/p(p-PDA) composites were evaluated by exposing them to CO2 gas and the changes in their conductivity were examined. The experimental setup used in the CO2 exposure of prepared materials was reported in the literature [3-, H2SO4-, and H3PO4-doped p(p-PDA) polymers and their GA composites of known conductivities were exposed to CO2 gas for 30 min at 100 mL/min flow rate and the variations in their conductivities in response to CO2 gas exposure were illustrated in 2 exposure. The extent of conductivity falls of these acid-doped P(p-PDA) polymers and GA/P(p-PDA) composites are given in −8 ± 1.12 × 10−8 S·cm−1 to 3.12 × 10−9 ± 7.57 × 10−10 S·cm−1 after 30 min of CO2 gas exposure. Similarly, HNO3- and H2SO4-doped P(p-PDA) polymers have experienced, respectively, about 13-, and 18-fold decrease after 30 min of CO2 gas exposure. On the other hand, the decrease in conductivity of H3PO4-doped P(p-PDA) was slightly lower than that of the others with approximately 6-fold reduction.Carbon dioxide  composites from 5.16 × 10−2 ± 2.72 × 10−3 S·cm−1 to 8.52 × 10−5 ± 1.21 × 10−5 S·cm−1 after 30 min of CO2 gas exposure were attained.Moreover, it was found that the conductivity of bare GA was calculated to be 2.17 × 102SO4-doped GA/p(p-PDA) composites appeared to show an approximately 100-fold decrease from 8.78 × 10−3 ± 1.17 × 10−3 to 8.91 × 10−5 ± 1.19 × 10−5 S·cm−1 after 30 min of CO2 gas exposure, whereas the changes in the conductivity of HNO3-, and H3PO4-doped GA/P(p-PDA) composites were far less than the others, with a 13- and 12-fold decrease, respectively, after 30 min exposure to CO2 gas.Additionally, the conductivity of H2 exposure can be explained by diffusion of the gas within aerogel networks, which were the main reason that more conductivity reduction has been observed in GA/p(p-PDA) composites as compared to those of the free-standing p(p-PDA) polymers. More specifically, the CO2 gas could not diffuse into the interiors of the bare p(p-PDA) pellets, yet as a result of much higher surface area and macroporous structure of GA/p(p-PDA) composites along with the gas adsorption properties of the graphite and graphene oxide layers. Consequently, larger amounts of the CO2 gas were able to diffuse towards the porous network of the HCl- and H2SO4-doped GA/p(p-PDA) composites and taped the pores much better. In addition, the positive charges, and unpaired electrons of in situ synthesized p(p-PDA) within GAs can easily interact with CO2 molecules, which have π-type C=O binding ability and a lone pair of electrons [The change in the conductivity of GA/p(p-PDA) composites in the course of COlectrons .2 molecules, and sensitivity can be defined as the ratio between the output signal (conductivity) per mole of the sensed molecule, CO2. Hence, the HCl-doped GA/p(p-PDA) composites exhibiting the greatest change in conductivity was studied for their sensitivity towards CO2 gas, and the change in the conductivity of GA/p(p-PDA)-HCl composites was recorded at different exposure time scales, 0.5, 1, 5, 10, 20, 30, 45, 60 min at 200 mL/min CO2 flow rate, and the corresponding CO2 sensitivity graph is presented in 2 exposed to GA/p(p-PDA)-HCl composites at 0.5, 1, 5, 10, 20, 30, 45 and 60 min periods were calculated as 0.0045, 0.009, 0.045, 0.09, 0.18, 0.27, 0.40, and 0.54 moles, respectively. It was observed that the change in conductivity of GA/p(p-PDA)-HCl composites were increased with the increase in the time of CO2 gas exposure. Accordingly, 5.7-, 26.4-, 128-, 358-, 511-, 605-, 629-, and 633-fold conductivity decreases were recorded for GA/p(p-PDA)-HCl composites upon exposure to 198, 396, 1980, 3960, 7920, 11,980, 17,820, and 23,960 ppm CO2, respectively. The required concentration of CO2 for a healthy breath was stated to be less than 1000 ppm, and the concentration of CO2 in the atmosphere was 417 ppm as of May 2020. According to the sensitivity graph of HCl-doped GA/p(p-PDA) composites, 193.6 ppm is enough to trigger a 5.7-fold decrease in the conductivity of GA/p(p-PDA)-HCl composites and gradually decreased e.g., a 26.4- and 128- fold decrease can be monitored at 397.2, and 1936 ppm CO2 concentration in only 1 and 5 min exposures, respectively. These results clearly indicate that GA/p(p-PDA)-HCl composites can be used as a promising candidate for CO2 detection in various environmental applications including the detection of CO2 in the atmosphere. In here, the GA/p(p-PDA)-HCl as CO2 sensor is based on the change in conductivity upon CO2 exposure in a concentration-dependent manner, so that the sensitivity can be calculated as conductivity changes (fold)mole of CO2. The sensitivity of GA/p(p-PDA)-HCl is calculated from the slope of 2 gas.The response of GA-based composites can be defined as a change in the conductivity in the presence of various amounts of CO−2 ± 2.72 × 10−3 S·cm−1 to 8.52 × 10−5 ± 1.21 × 10−5 S·cm−1 after 30 min of CO2 gas exposure, and was recovered to 4.97 × 10−2 ± 1.13 × 10−3 S·cm−1 after 2 h incubation at 60 °C. The conductivity of GA/p(p-PDA)-HCl composites decreased from 4.97 × 10−2 ± 1.13 × 10−3 S·cm−1 to 8.54 × 10−5 ± 1.11 × 10−5 S·cm−1 after the 2nd cycle of CO2 gas exposure, and was recovered back to 4.51 × 10−2 ± 9.35 × 10−4 S·cm−1 after the 2nd cycle. Then, the conductivity of the composites decreased to 7.87 × 10−5 ± 9.84 × 10−6 S·cm−1 after the 3rd CO2 gas exposure, and it was again increased to 3.91 × 10−2 ± 8.11 × 10−4 S·cm−1 after the 3rd regeneration. The conductivity of GA/p(p-PDA)-HCl composites decreased from 3.91 × 10−2 ± 8.11 × 10−4 S·cm−1 to 7.11 × 10−5 ± 1.71 × 10−5 S·cm−1 in the 4th cycle of CO2 exposure, and then restored to 3.13 × 10−2 ± 4.01 × 10−4 S·cm−1 after the 4th recovery. Finally, the conductivity decreased to 6.32 × 10−5 ± 9.23 × 10−6 S·cm−1 after the 5th 30 min of CO2 gas exposure. These findings clearly demonstrated that the GA/p(p-PDA)-HCl composites can be used in the detection of CO2 gas repeatedly after a 2 h of incubation at 60 °C, with almost full recovery to its initial conductivity measuring capability.Furthermore, the reusability of the prepared GA/p(p-PDA)-HCl composites were investigated for five successive (re)use and regeneration cycles, and the corresponding graph is illustrated in 3, H2SO4, and H3PO4 used in doping of p(p-PDA) polymers were found to have a considerable influence on the electrical conductivity of both p(p-PDA) polymers and their corresponding GA-based composites. GA/p(p-PDA) composites achieved higher electrical conductivities than both their bare p(p-PDA) polymers and bare GAs. The in situ synthesis of GA/p(p-PDA)-HCl composites gave rise to a 250-fold increase in the conductivity of bare GAs from 2.17 × 10−4 ± 3.15 × 10−5 S·cm−1 to 5.16 × 10−2 ± 2.72 × 10−3 S·cm−1. Moreover, GA/P(p-PDA)-HCl composites were revealed to show the best response to CO2 gas amongst other acid-doped composites, with an approximately 600-fold conductivity decrease. Owing to the well-known properties of graphene oxides and GAs to absorb gases, and the response of in situ prepared p(p-PDA) conductive polymer, GA/p(p-PDA) composites were shown to be a sensor for CO2 gas with the capability of measuring CO2 levels in the atmosphere, which are well within its sensitivity level. Although the gravimetric amount of in situ synthesized p(p-PDA) polymers with HCl doping in GA/p(p-PDA) composites is less than that of H2SO4- and H3PO4-doped GA/p(p-PDA) composites, better electrical conductivity, as well as better CO2 response of this material, affords better outcomes in terms of real applications. Furthermore, GA/p(p-PDA)-HCl composites exhibited a 5.7-fold conductivity decrease even at 193.6 ppm CO2 gas concentration in only 30 sec, and a 26.4-fold decrease in 1 min at 397.2 ppm CO2 concentration, along with the sensitivity of 1288.7 fold conductivity decrease/mole of CO2 gas. Since these concentrations are less than the half of those reported as dangerous, GA/p(p-PDA)-HCl composites hold remarkable potential in the determination CO2 as a sensor in many industrial effluents and mining applications, and so on.Herein, we demonstrated the potential usage of GAs as templates and sensory materials. Using GAs as templates in the in situ synthesis of conductive p(p-PDA) polymers clearly reveals the the versatility of these materials. The acid types, HCl, HNO2 sensors and detectors in chimneys of factories, dwellings, and lab gadgets such as CO2 incubators, etc., to determine the level of emitted/generated or released CO2, but they may also be useful to track accumulated levels of CO2 in different milieus, including climate chambers and indoor living environments, and so on.Overall, it is apparent that GA-based materials offer a lot of potential, as they are not restricted to CO


Streptomyces aminophilus subsp. notonesogenes 647-AV1 and S. aminophilus DSM 40186. In a high throughput antifungal screening campaign, we identified an alternative notonesomycin A producing strain, Streptomyces sp. A793, and its biosynthetic gene cluster. From this strain, we further characterized a new more potent antifungal non-sulfated analogue, named notonesomycin B. Through CRISPR–Cas9 engineering of the biosynthetic gene cluster, we were able to increase the production yield of notonesomycin B by up to 18-fold as well as generate a strain that exclusively produces this analogue.Notonesomycin A is a 32-membered bioactive glycosylated macrolactone known to be produced by  Natural products (NPs) represent a diverse source of bioactive compounds with clinical relevance, as evidenced by the fact that half of current drugs are NP or NP-derived , 2. TheyCandida species are common pathogens associated with invasive fungal diseases contributing to high disease burden + peak at m/z 1454 and the HR-ESI-MS measurement established the molecular formula as C68H111NO30S. The 13C NMR data , and eight methyl  carbon atoms. The 1H NMR spectrum , 6.58 , 7.68 ], indicating a tri-substituted aromatic ring system. The remaining two olefinic proton signals resonated at δ 5.95 and 7.04 with a large coupling constant of 15 Hz suggesting a E configuration and were assigned to an α, β-unsaturated ester group based on the observed δH 5.95 (H-2) and 7.04 (H-3) to δC 168.4 (C-1) HMBC correlations , 0.84 , 0.90 , 0.96 , 0.97 , and 1.06 , 1.27 , and 1.32  and were assigned to H-44, H-42, H-43, H-40, H-39, H-41, H-6ʹʹʹ and H-6ʹ, respectively. The two oxymethines observed at δH 2.79 (δC 63.8) and 2.73 (δC − 60.3) were characteristic of an epoxide moiety with cis-orientation based on the observed small coupling constant of 2 Hz + peak at m/z 1374 and the HR-ESI-MS measurement established the molecular formula as C68H111NO27. The difference in 80 mass units as compared to 1 suggested a loss of a [SO3] group. The 1H, 13C, COSY, HSQC and HMBC NMR spectra , δ 5.11 ], an epoxide , a trisubstituted benzene ring , a macrolide incorporating a hemiketal ring, and an α, β-unsaturated lactone moiety  similar to those of 1. The 1D and 2D NMR , C-17  and C-18  of the 6-membered hemiketal moiety embedded within the macrocyclic ring. Since there was no change in the total number of carbon and protons in 2 as compared to 1, the observed 80 mass unit loss in HRMS data indicated the presence of a hydroxyl group instead of the sulfate group at C-17. This was further supported by the observed 8.2 ppm upfield shift for C-17 in 2  + ; 1H and 13C NMR data, see Additional file Brownish amorphous solid; [α]d 307 3.9 nm; HR-ED − 56 ; UV (MeOH) λmax (log ε) 229 sh (3.94) and 309 (3.89) nm; HR-ESI-MS m/z 1374.7414 [M+H]+ ;1H and 13C NMR data, see Additional file White amorphous solid; [α]D − 56 c .3, MeOH;1H, G-COSY, multiplicity-edited G-HSQC, and G-HMBC spectra) or BBO (13C spectra) probe heads equipped with z-gradients. Spectra were calibrated to residual protonated solvent signals . Preparative HPLC analysis was performed on the Agilent 1260 Infinity Preparative-Scale LC/MS Purification System, completed with Agilent 6130B single quadrupole mass spectrometer for LC and LC/MS Systems. The samples were separated on an Agilent Prep C18 column (100 × 30 mm) by gradient elution with a mixture of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The HR-ESI-MS spectra were acquired on Agilent UHPLC 1290 Infinity coupled to Agilent 6540 accurate-mass quadrupole time-of-flight (QTOF) mass spectrometer equipped with a splitter and an ESI source. The analysis was performed with a C18 4.6 × 75 mm, 2.7 µm column at flowrate of 2 mL/min, under standard gradient condition of 0.1% formic acid in water and 0.1% formic acid in acetonitrile over 15 min.Optical rotations were recorded on a JASCO P-2000 digital polarimeter. UV spectra were obtained on a GE Healthcare Ultrospec 9000 spectrophotometer. NMR spectra were collected on a Bruker DRX-400 NMR spectrometer with Cryoprobe, using 5-mm BBI  at 5 × 105 cells mL−1. The cells were incubated with the compounds at 35 °C for 24 h  and 48 h (A. fumigatus); and 37 °C for 24 h for both bacteria. The effect of the compounds on bacterial growth was evaluated by measuring the optical density at 600 nm using a microplate reader. Mammalian cell cytotoxicity was assessed with A549 human lung carcinoma cells (ATCC® CCL-185™) seeded at 1500 cells well−1, while both PANC-1 human pancreatic carcinoma cells (ATCC® CRL-1469™) and HepG2 human hepatocellular carcinoma cells (ATCC® HB-8065™) which were both seeded at 2500 cells well−1. These cells were treated with the compounds for 72 h at 37 °C in the presence of 5% CO2. PrestoBlue™ cell viability reagent (Life Technologies) was used to assess the cytotoxic effect of the compounds. Microplates were incubated with this dye for 2 h before measuring the fluorescence at excitation 560 nm and emission 590 nm. All assays were performed in triplicate on two different test runs.Minimum inhibition concentration (MIC) was determined using the microbroth dilution method according to guidelines of the Clinical Laboratory Standards Institute (CLSI) with slight modifications. Assays were carried out with Streptomyces sp. A793 with modified CTAB method [−1) and 15 µL of RNase (10 mg mL−1) at 37 °C overnight. Subsequent procedures of the CTAB method were carried out, followed by phenol–chloroform purification. An additional purification step was carried out using Mag-Bind® RxnPure Plus (OMEGA bio-tek) as per manufacturer’s protocol.High-quality and molecular weight genomic DNA (> 20 kb) was extracted from B method . Cells wGenome sequencing was performed with the Pacific Biosciences (PacBio) RSII. Purified genomic DNA was sheared to approximately 20 kb using a g-Tube (Covaris). A SMRTbell library was prepared according to manufacturer’s instructions, loaded with a MagBead bound library protocol onto two SMRTCells at 0.125 and 0.3 nM, and sequenced using the P5-C3 chemistry on the PacBio RSII instrument (Pacific Biosciences) with a 180 min movie time. De novo assembly was performed with the Hierarchical Genome Assembly Process 3 (HGAP3)  in the SS. armeniacus strain JCM 3070 (accession numbers NR_112046 and NR_112047) covering a length of 1530 nucleotides. In addition, we found a 100% identity match of a 1330 nucleotide sequence stretch of the A793 strains’ 16S rRNAs to the 16S rRNA partial sequence available in the draft genome of S. armeniacus strain ATCC 15676 . This eThe identification of open reading frames (ORFs) was obtained with an in-house pipeline that takes into account the gene predictions by Prodigal 2.6.2 , GlimmerE. coli DH5α or OmniMAX™ (Thermo Fisher). Primers used in this study are listed in Additional file BbsI-mediated Golden Gate assembly before the respective homology flanks were inserted via Gibson assembly. The protocol for plasmid construction was previously described [All DNA manipulations were carried out in escribed .E. coli strains and colonies were picked into LB with 50 mg/L apramycin (Sigma). A diaminopimelic acid (DAP) negative ET12567/pUZ8002 donor strain was used [600 of 0.4–0.6. 400 µL of the culture was pelleted, washed twice and resuspended in LB without apramycin. The washed E. coli cells were then mixed with spores at 1:5 volume ratio and spotted on R2 without sucrose plates. After incubation for 16–20 h at 30 °C, the plates were flooded with nalidixic acid and apramycin and incubated until exconjugants appear. Exconjugants were streaked onto MGY plates containing apramycin at 30 °C followed by re-streaking to MGY plates at 37 °C to cure the CRISPR–Cas9 plasmid containing a temperature-sensitive origin of replication. Apramycin-sensitive clones growing at 37 °C were then subjected to validation of promoter knock-in and genome editing as described below.Promoter knock-in constructs are transformed into conjugating was used . OvernigGenomic DNA from wild type and exconjugants from the indicated strains were isolated from liquid cultures using the Blood and Tissue DNeasy kit (Qiagen) after pretreating the cells with 20 mg/mL lysozyme for 30 min at 37 °C. PCR was performed using control primers beyond the homology regions or knock-in specific primers  after 9 days growth and treated with TURBO™ DNase (Ambion). The quality of purified RNA was determined on an Agilent 2100 bioanalyzer . DNA free RNA samples were used for first strand cDNA synthesis with random hexamers using iScript™ (Biorad) reverse transcription supermix. RT-qPCR was performed on Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System using SYBR FAST qPCR master mix (KAPA). Analysis was carried out with QuantStudio™ Real-Time PCR Software, Version 1.3. The relative abundance of transcripts was calculated by delta-delta CT relative quantitation method/comparative threshold cycle (CT) method [rpoB, hrdB, recA and atpB genes of Streptomyces sp. A793 was used as endogenous reference to normalize gene expression of each target gene  method . The avem/z 121.0509 and HP-921 ion at m/z 922.0098.A 10 mL culture was freeze dried and extracted with equal volume of methanol and sonicated for 10 min. A wet equivalent of the extract was centrifuged and injected into LC-QTOF for analysis. The HRESIMS spectra were acquired on Agilent UHPLC 1290 Infinity coupled to Agilent 6540 accurate-mass quadrupole time-of-flight (QTOF) mass spectrometer equipped with a splitter and an ESI source. The analysis was performed with a C18 4.6 × 75 mm, 2.7 µm column at flowrate of 2 mL/min, under standard gradient condition of 100% water with 0.1% formic acid to 100% acetonitrile with 0.1% formic acid over 15 min. The typical QTOF operating parameters were as follows: positive ionization mode; sheath gas nitrogen, 12 L/min at 295 °C; drying gas nitrogen flow, 8 L/min at 275 °C; nebulizer pressure, 30 psig; nozzle voltage, 1.5 kV; capillary voltage, 4 kV. Lock masses in positive ion mode: purine ion at Pairwise comparisons between means abundances of the compounds detected from the mutant and wild type strains were carried out using a Student t-test  where the null hypothesis states that the means are equal. The difference between the mutant and wild type strains is considered statistically significant if the Student t-test gives a P value < 0.05.Additional file 1: Table S1. NMR spectral dataa of notonesomycin A (1) and notonesomycin B (2). Table S2. Annotation of 59 ORFs of the notonesomycin BGC by sequence homology searches using BLASTp against the NCBI nr database. Table S3. Media compositions used in shake flask fermentation. Table S4. Oligos used in this study. Table S5. List of primers used for RT-qPCR. Figure S1. Structure and gross structures of notonesomycins. Selected HMBC correlations of 1 and 2. Figure S2.13C NMR spectrum  of Notonesomycin A (1). Figure S3.1H NMR spectrum  of Notonesomycin A (1). Figure S4. COSY spectrum  of Notonesomycin A (1). Figure S5. HSQC spectrum  of Notonesomycin A (1). Figure S6. HMBC spectrum  of Notonesomycin A (1). Figure S7.13C NMR spectrum  of Notonesomycin B (2). Figure S8.1H NMR spectrum  of Notonesomycin B (2). Figure S9. COSY spectrum  of Notonesomycin B (2). Figure S10. HSQC spectrum  of Notonesomycin B (2). Figure S11. HMBC spectrum  of Notonesomycin B (2). Figure S12. Phylogenetic analysis of 59 acyltransferases (AT) amino acid sequences from notonesomycin (Ntc AT), brasilinolides (Nbr AT), PM100117 and PM100118 (GonP AT) BGCs. Figure S13. Proposed biosynthesis pathways for A) 4-amino 3-hydroxybenzoic acid and B) deoxysugars. Figure S14. Sequence analysis of glycosyltransferases Nbc18 and Nbc22 from the notonesomycin BGC with known glycosyltransferases. Figure S15. Sequence analysis of cytochrome P450 enzymes  from the notonesomycin BGC with other cytochrome P450 with either hydroxylation or epoxidation function. Figure S16. Sequence analysis of methyltransferases Nbc1 and Nbc56 from Streptomyces sp. A793 present in the notonesomycin BGC with other known O-, N- and C-methyltransferases. Figure S17. LC–MS spectra  for wild type and nbc20 and nbc21 double KO mutant. Figure S18. Sequence analysis of sulfotransferase Nbc48 from the notonesomycin BGC with sulfotransferases that have been associated with other BGCs.


Scientific Reports 10.1038/s41598-019-40332-z, published online 04 March 2019Correction to: This Article contains an incorrect version of Figure 8, where the trend lines did not consider the varying element size in the radial and vertical dimensions during averaging.Furthermore, in Figure 8, there is an error in the unit for panels (b) and (c).As a result, Figure 8 legend,a) Vertical Mass flux in kg/s (b) Inertial stability, I2 (s−1) (c) Static stability, N2 (d) Generation of available potential energy (m2s−3) (e) Conversion from potential to kinetic energy (m2s−3).”“Time-series plots  for Phailin within the RMW. (should read:a) Vertical Mass flux in kg/s (b) Inertial stability, I2 (s−2) (c) Static stability, N2 (s−2) (d) Generation of available potential energy (m2s−3) (e) Conversion from potential to kinetic energy (m2s−3).”“Time-series plots  for Phailin within the RMW. (The correct Figure 8 appears below as Figure


In the title compound, intermolecular C—H⋯π interactions and π–π stacking interactions help to stabilize the crystal structure, forming a three-dimensional network. 22H33N3O4, crystallizes in the triclinic space group PThe title compound, C The experiments were performed in a wide temperature range (from room temperature to 413 K) and led to multicomponent mixtures of products at elevated temperatures, from which we were unable to isolate the target adduct 5.This work is a continuation of the study of Diels–Alder reactions on bis-diene systems, which was previously carried out on the example of the tandem [4 + 2]/[4 + 2] cycloaddition between bis-furyl dienes similar to et al., 2016et al., 2017abet al., 2017et al., 2018et al., 2017et al., 2010abet al., 20144.However, taking into account the importance of the non-covalent bond-donor/acceptor properties of the nitrogen atom in N-heterocycles for synthesis, catalysis and the design of new materials °. The C6—N2—C17—C18 and C7—N2—C17—C18, C5—C6—N2—C17, C8—C7—N2—C17 and C6—N2—C7—C8 torsion angles are −163.52 (15), 71.9 (2), −87.35 (17), −155.20 (14) and 80.67 (16)°, respectively. All of the bond lengths and angles in the title compound 4 are of usual values.As shown in Fig. 24 is defined by π–π stacking  and C—H⋯π  interactions, forming a three-dimensional network  to 1.6027 (blue) a.u. is shown in Fig. 4The Hirshfeld surface analysis bis(methylene)[bis(1H-pyrrole-1-carboxylate)] skeleton revealed 37 structures similar to the title compound 4. Only three of them are closely related to the title compound, viz. di-tert-butyl 2,2′-bis(1H-pyrrole-1-carboxylate) in the space group P21/n -1H-pyrrol-2-yl]-2,5-bis phenyl}-1H-pyrrole-1-carboxylate in the space group C2/c -5-(methoxycarbonyl)-pyrrol-2-yl)methane in the space group PA search of the Cambridge Structural Database  and C—H⋯π interactions were found, which could help to rigidify the molecular conformation. In NANLAP, the dihedral angle between the two pyrrole ring is 82.77°.vs aliphatic, large vs small) may account for the distinct intermolecular interactions in the crystals.In the three structures closely related to the title compound, the different linkers between the two pyrrole units (aromatic Di-tert-butyl dicarbonate  was added to a solution of N,N-bis(1H-pyrrol-2-ylmethyl)ethanamine  and DMAP  in CH3CN (50 mL) at room temperature under an argon atmosphere. The mixture was stirred for 6 h at room temperature. The reaction mixture was poured into a 5% solution of NH3 in H2O (300 mL) and extracted with CH2Cl2 (3 × 50 mL). The combined organic layers were dried over MgSO4, filtered and concentrated. Flash chromatography purification on aluminium oxide (hexane) of the residue yielded the title compound as colourless crystals. Single crystals suitable for X-ray diffraction analysis were obtained by slow evaporation of an EtOAc/hexane solution at room temperature. Colourless prisms. Yield 14.25 g (60%). M.p. = 349.8–351.5 K . IR (KBr), ν (cm−1): 3112, 3172. 1H NMR : δ = 1.08 , 1.57 , 2.67 , 3.90 , 6.09 , 6.31 , 7.16 . 13C NMR : δ = 12.6 (NCH2CH3), 28.1 , 48.9 (N–CH2–CH3), 52.8 , 83.3 , 110.2 , 111.7 , 120.9 , 134.9 , 149.5 . Elemental analysis calculated for C22H33N3O4 (%): C 65.12, H 7.88, N 10.73; found (%): C 65.48, H 8.24, N 10.41.Uiso(H) = 1.2 or 1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989020014966/yz2002sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020014966/yz2002Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020014966/yz2002Isup3.cmlSupporting information file. DOI: 2043609CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Both  N-donor pyridyl ligands are reported, namely, bisoxido(pyridine-κN)vanadium(IV), [V(C5H7O2)2O(C5H5N)], 1, bisoxido(pyridine-4-carbonitrile-κN)vanadium(IV), [V(C5H7O2)2O(C6H4N2)], 2, and bis(4-methoxypyridine-κN)oxidovanadium(IV), [V(C5H7O2)2O(C6H7NO)], 3, Compounds 1–3 have the formulae VO(C5H7O2)2L, where L = pyridine (1), 4-cyano-pyridine (2), and 4-methoxypyridine (3). Compound 1 was previously reported  and redetermined here at cryogenic temperatures. Compounds 1 and 2 as pyridine and 4-cyanopyridine adducts, respectively, crystallize as distorted octahedral structures with the oxo and pyridyl ligands trans to one another. A crystallographic twofold axis runs through the O—V—N bonds. Compound 3 containing a 4-methoxypyridine ligand crystallizes as a distorted octahedral structure with the oxo and pyridyl ligands cis to one other, removing the twofold symmetry seen in the other complexes.Crystal structures for a series of bis(acetylacetonato)oxovanadium(IV) complexes containing  al. 1984. Kexue T The complexes were synthesized rapidly in an Anton Paar Monowave 50 synthesis reactor in 5 minutes at 323 K and crystallized upon cooling the mother liquor.Oxovanadium complexes chelated by two acetylacetonate ligands form a five-coordinate bonding system that can act as a Lewis acid ° and 99.84 (3)° and in 2 are 98.42 (4)° and 98.91 (3)°.Figs. 13 exists as a different isomeric form, with the oxo and 4-methoxypyridine ligand being cis to one another. This removes the twofold symmetry seen in compounds 1 and 2 and compound 3 crystallizes in the space group P21/n. Similarly to 1 and 2, compound 3 adopts a distorted octahedral geometry upon chelation by two bidentate acetylacetonate ligands.Compound 1–3 are are similar to those observed in related complexes  and 2.4022 (15) Å, respectively. However in 3, the V—N bond length is much shorter at 2.1140 (12) Å, likely from a combination of the cis-isomeric structure in 3 and the electron-donating methoxy group of the 4-methoxypyridine ligand.The V—O and V=O bond lengths for 1–3. Figs. 41, these interactions are centered around the oxo ligand, with methyl groups of the acetylacetonate ligands forming CH2—H⋯Oii interactions at a distance of 2.575 (9) Å between O1ii and H5A and the aryl protons of the pyridine ligand forming Ar—H⋯Oiii interactions at a distance of 2.429 (9) Å between O1iii and H7 . Similar interactions exist in 2 with CH2—H⋯Oii interactions at a distance of 2.591 (9) Å between the oxo ligand, O1ii, and H4C of the methyl group of the acetylacetonate and Ar—H⋯Oiii interactions at a distance of 2.588 (9) Å between the 4-cyanopyridine proton H7 and O1iii . Compound 2 also displays interactions between the methyl groups of the acetylacetonate and the π-bond within 4-cyanopyridine at a distance of 2.682 (9) Å from the proton to the center of the π-bond.Several non-covalent interactions Tables 1–3 ▸ ▸ e1 and 2, compound 3 displays different types of non-covalent interactions. The methine proton, H2, of the acetylacetonate interacts with the oxo ligand oxygen, O1i at a distance of 2.568 (8) Å and the aryl protons H12 and H14 interact with acetylacetonate oxygens atoms O2ii and O4iii at distances of 2.366 (8) Å and 2.569 (8) Å, respectively . Additionally, there are weaker interactions between the π-system of the acetylacetonate and 4-methoxypyridine, evident by a 3.196 (9) Å distance from C3 to C14iii.Compared to 2) and the N-donor ligands pyridine, 4-cyanopyridine, and 4-methoxypyridine were purchased and used without further purification. To an Anton Paar Monowave synthesis reactor vial, a 1:1 molar ratio of VO(acac)2 and an N-donor ligand  was added and dissolved into 5 mL of dichloromethane. Once dissolved completely, each solution was reacted in an Anton Paar Monowave 50 synthesis reactor at 323 K for 5 min. Following all of the reactions, a slight precipitate was filtered and the resulting filtrate was allowed to slowly evaporate to produce single crystals suitable for X-ray diffraction studies. In addition to characterization by single crystal X-ray diffraction, each complex was characterized by FTIR spectroscopy. Compound 1 IR (neat) ν (cm−1): 3065(w), 2964(w), 1574(m), 1522(s), 1443(m), 1378(s) 1351(s), 1274(m), 1218(w), 1196(w), 1147(w), 1074(w), 1018(m), 964(s), 931(m), 890(w), 782(w), 763(m), 708(m), 676(m). Compound 2 IR (neat) ν(cm−1): 3084(w), 3034(w), 1557(m), 1540(w), 1522(s), 1411(m), 1375(s), 1277(m), 1211(w), 1190(w), 1018(m), 960(s), 929(m), 850(m), 789(m), 737(w), 679(m), 667(m). Compound 3 IR (neat) ν(cm−1): 3072(w), 3017(w), 1577(m), 1513(s), 1431(m), 1367(s), 1329(m), 1291(m), 1273(m), 1210(m), 1109(w), 1058(w), 1029(m), 948(s), 928(m), 836(m), 807(m), 780(m), 678(w), 658(m).Bis(acetylacetonato)oxovanadium(IV) (VO(acac)I ≥ 10σ(I). All data were corrected for Lorentz and polarization effects, and runs were scaled using SADABS  for methyl and 1.2Ueq(C) for all other hydrogen atoms.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989020006246/zl2781sup1.cifCrystal structure: contains datablock(s) compound1, compound2, compound3, global. DOI: 10.1107/S2056989020006246/zl2781compound1sup2.hklStructure factors: contains datablock(s) compound1. DOI: 10.1107/S2056989020006246/zl2781compound2sup3.hklStructure factors: contains datablock(s) compound2. DOI: 10.1107/S2056989020006246/zl2781compound3sup4.hklStructure factors: contains datablock(s) compound3. DOI: 2002530, 2002529, 2002528CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


These effects were inhibited by knocking down the Uqcrb and Ndufs6 genes of mitochondrial respiratory chain, whose transcriptions were regulated by candidate magnetoreceptor genes Isca1 and Cry1/Cry2. SMF exposure also promoted CD8+ T cell granule and cytokine secretion and repressed tumor growth in vivo. SMFs enhanced CD8+ T cell cytotoxicity, and the adoptive transfer into tumor-bearing mice resulted in enhanced antitumor effects. Collectively, our study suggests that moderate SMFs enhance CD8+ T cell cytotoxicity by promoting mitochondrial respiration and promoted the antitumor function of CD8+ T cells.With the discovery of magnetoreceptor mechanisms in animals, it materialized the novel applications of controlling cell and animal behaviors using magnetic fields. T cells have shown to be sensitive to magnetic fields. Here, we reported that exposure to moderate SMFs (static magnetic fields) led to increased granule and cytokine secretion as well as ATP production and mitochondrial respiration from CD8 CD4+ T cells are also called helper T cells, which support the activity of other immune cells by releasing T cell cytokines such as IFNγ, IL-4, IL-17 and TNFα2. CD8+ T cells are also called cytotoxic T lymphocytes (CTLs), which mediate the direct killing of infected, damaged, and dysfunctional cells and play critical roles in antitumor immunity3. The cytolytic targeted killing of cancer cells by CTLs requires perforin-mediated release of granzymes, mainly granzyme B, from cytotoxic granules4. In addition, CTLs can eliminate cancer cells or virally infected cells by secreting cytokines such as IFNγ and TNFα5.T cells are critical components of the adaptive immune system. T cells can be divided into CD46. Many animals sense geomagnetic field (25 − 65 μT) for the purpose of orientation or to navigate and migrate over long distances7. Cryptochromes (Cry) have been reported to be the best biochemical magnetoreceptor candidates in animals such as birds and Drosophila, which mediate light-dependent magnetosensitivity via a radical-pair reaction10. Another magnetoreceptor candidate, an iron-sulfur cluster assembly protein 1 (Isca1), has been reported to form a rod-like complex with Cry and works like a biocompass11.The magnetic field is an important physical factor similar to the temperature and pressure existing in the environment. Depending on intensity, static magnetic fields (SMFs) can be classified as weak (< 1 mT), moderate (1 mT–1 T), strong (1–5 T), and ultrastrong (> 5 T)16. It seems that strong and ultrastrong SMFs have harmful effects on normal cells, such as altering cleavage planes, changing the orientation and morphology of mitotic spindles and causing DNA damage18. However, moderate SMFs showed much fewer harmful effects on normal cells and exhibited great potential in changing cell behaviors, including altering stem cell proliferation, promoting osteoclast differentiation, impairing cancer cell proliferation, enhancing oligodendrocyte differentiation and promoting neurotrophic factor secretion19. With the discovery of magnetoreceptor genes and related mechanism models21, it materialized the novel applications of controlling cell and animal behaviors based on magnetoreceptor mechanisms.In addition, a lot of recent studies have shown that SMFs have various cellular effects on cells22. Moreover, exposure to 1.5 T SMFs leads to suppress the release of cytokines IL-6, IL-8, and TNF-α, but assists the production of IL-10 of human lymphocytes and macrophages23. These results suggest that strong SMFs can change the behavior of T cells. However, the underlined mechanism of SMFs influencing T cell behavior is largely unknown, and lacks in vivo evidence.T cells have presented to be sensitive to SMFs. Exposure to strong SMFs (> 3 T) has been reported to induce genotoxic effects and increase apoptosis of human T lymphocytes+ T cells as well as elevated ATP production and mitochondrial respiration. These effects were inhibited by knocking down the Uqcrb or Ndufs6 genes, which are related to mitochondrial respiratory electron transport chain. In addition, knocking down the magnetoreceptor genes Isca1 or Cry1/Cry2 led to reduced transcription of Uqcrb and Ndufs6, and also reversed the increased granule and cytokine secretion and ATP production of SMF treated CD8+ T cells. Moreover, SMFs enhanced CD8+ T cell cytotoxicity, and adoptive transfer of SMF-treated CTLs into tumor-bearing mice resulted in enhanced antitumor effects. Taken together, our study not only elucidate the molecular mechanism of SMFs in enhancing CD8+ T cell cytotoxicity by promoting mitochondrial metabolism, but also provides a new approach of promoting the antitumor function of CD8+ T cells using a physical method.In the present study, we studied the cellular effects of moderate SMFs on T cells using magnetic intensity of 0.3 T and 0.6 T in mouse model. We found that exposure to moderate SMFs led to increased granule and cytokine secretion from CD8+ T cells or CD8+ T cells from the spleens of C57/B6 mice were cultured in plates that were placed on permanent magnets with a magnetic intensity of 0.3 T or 0.6 T  was higher in 0.3 T SMF-treated cells than in control cells at all time points  stimulation at different time points. Purified CD4+ T cells and CD8+ T cells. The cytokine production of purified CD4+ T cells showed no significant changes between SMF-treated cells and control cells at all time points  and the sham group . Two main clusters of genes with a fold change ≥ 2 were identified by functional enrichment analysis. Cluster 1 contained 198 genes that mainly participated in mRNA metabolic processes and RNA processing  in CD8+ T cells. The OCR was also elevated in SMF-treated CD8+ T cells compared with that in control cells  and nonpulsed splenocytes  were used as target cells and intravenously coinjected at a 1:1 ratio into recipient mice. Non-pulsed splenocytes were not lysed and used as internal controls. The cytotoxicity was calculated as [1 − (% CFSElow)/(% CFSEhigh)] × 100%28. Compared to the control CTLs, the SMF-treated CTLs killed more OVA257–264-pulsed splenocytes in vivo, and the flow cytometry data showed that 28.5% versus 33.9% CFSElow-target cells left  or CFSEhigh (unplused with peptide) was approaching 50%  and treated them with or without SMFs to assess antitumor function. Resin fiber plates embedded with 0.3 T or 0.6 T button magnets were used to expose mice to the SMFs  and effector memory population (CD62LlowCD44high) of both CD4+ T and CD8+ T cells was also comparable -T cell therapies, both of which can redirect T cells to more efficiently target cancer antigens16. Various experimental parameters, such as cell types, magnetic field parameters (including magnetic field intensity and direction), and exposure time, often led to different cellular effects. In our study, we screened the cellular effects of moderate SMFs on the function of both CD4+ T and CD8+ T cells at various time points after TCR stimulation and upon treatment with SMFs at different intensities. We observed that exposure to moderate SMFs enhanced granule and cytokine secretion of CD8+ T cells at 72 h after TCR stimulation but showed no obvious effects at 24 h and 48 h after stimulation. Moreover, SMFs did not affect cytokine secretion of CD4+ T cells at any of the time points. In addition, our in vivo studies also revealed that exposure to moderate SMFs promoted granule and cytokine secretion from tumor-infiltrating CD8+ T cells from PyMT mice but did not affect cytokine secretion from CD4+ T cells. These results suggested that SMFs showed highly variable effects between CD4+ T cells and CD8+ T cells. Considering the diversify and plasticity of the immune system, the biological effects of SMFs on different subsets of immune cells with various intensities or stimulation times are still worth to be further systematically studied.In recent years, the cellular effects of SMFs have been widely studiedC57BL/6 mice were purchased from SLAC. OT-I TCR transgenic mice and PyMT transgenic mice were obtained from The Jackson Laboratory. All mice were maintained in a specific pathogen-free facility and were genotyped by PCR. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences). We confirmed that all experiments were performed in accordance with relevant guidelines and regulations.l-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (10 U/ml). EL-4 cells were cultured in RPMI 1,640 medium supplemented with 10% fetal bovine serum, l-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (10 U/ml).B16-F10-OVA and EL-4 cell lines were kindly provided by Prof. Chenqi Xu (Chinese Academy of Sciences). B16-F10 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, Permanent magnets with an intensity of 0.3 T and 0.6 T were purchased from the Hangzhou Permanent Magnet Group Co., Ltd. The size of each 0.3 T permanent magnet is 10 cm × 10 cm × 5 cm, and the size of the 0.6 T permanent magnets is 10 cm × 10 cm × 10 cm. The magnetic intensity of the permanent magnets slowly attenuates with a distance of approximately 1 mm corresponding to a decrease in 5 mT. The resin fiber plates embedded with or without 0.3 T or 0.6 T button neodymium magnets were provided by Heye Health Technology Co., Ltd. of Zhejiang Province. The diameter of each button magnet is 1 cm, and the distance between any two button magnets is 2.5 cm. The magnetic intensity of these button magnets rapidly attenuates with a distance of approximately 1 mm corresponding to a decrease in 50 mT. The size of these resin fiber plates (32 cm × 12.5 cm × 1.5 cm) were designed to fit the mouse cages and were placed at the bottom of the cages. All the permanent magnets and magnetic plates were placed with the N pole up and the S pole down. The magnetic field strength was measured by KANETEC TESLA METER MODEL TM-801.Anti-CD4 (RM4-5), anti-CD8a (53-6.7), anti-CD44 (IM-7), anti-CD62L (MEL-14), anti-CD69 (H1.2F3), anti-Ly6G (Gr1) (RB6-8C5), anti-CD11b (M1/70), anti-TNFα (MP6-XT22), anti-IL-2 (JES6-5H4), and anti-IL-4 were purchased from BD Pharmingen. Anti-mFoxp3 (FJK-16 s), anti-granzyme B (NGZB) and anti-IL-17 (eBio17B7) were purchased from eBioscience. Anti-CD25 (PC61), anti-IFNγ (XMG1.2), and FITC Annexin V reagents were purchased from Biolegend. Anti-CD3 (145-2C11) and anti-CD28 (37.51) antibodies used for in vitro stimulation were obtained from BD Pharmingen. Brefeldin A was obtained from eBioscience, PMA and ionomycin were obtained from Merck. Collagenase IV used for tumor tissue digestion was purchased from Sigma.+ T cells or CD8+ T cells were isolated from the splenocytes of C57BL/6 mice using a CD4+ or CD8+ T cell negative selection kit (Stem Cell) and were stimulated with 1 μg ml−1 plate-bound anti-CD3 and anti-CD28 antibodies using 48-well cell culture plates. On the first day of stimulation, cell samples were seeded at 7 × 105 cells per well, then subculture at 1:2 ratio at 48 h after stimulation. Cell culture plates were placed on magnets once stimulated, and control cells were treated in the absence of magnets. To analyze the tumor-infiltrating T cells, tumors were first digested with collagenase IV, and tumor-infiltrating leukocytes were isolated by 40–80% (v/v) Percoll (GE) gradient centrifugation as previously described40. To measure the cytokine production of CD4+ or CD8+ T cells, the isolated cells were restimulated with PMA (50 ng ml−1) plus ionomycin (1 μM) for 4 h in the presence of 5 μg ml−1 brefeldin A and then stained with antibodies. Intracellular staining was performed after 10 min of fixation  at room temperature and 5 min of permeabilization in IC staining buffer  at 4 °C. Unstimulated T cells or T cells stained with isotype control antibody were used as negative controls. Cell fluorescence was determined using a two-laser FACSCalibur (BD Biosciences) flow cytometer, and data were analyzed with FlowJo software .CD4+ T cell cytotoxicity assay was performed as previously described41. Purified CD8+ T cells from OT-I mice were stimulated with anti-CD3/anti-CD28 mAbs in vitro for 4 days (96 h) as effector CTL cells, which were treated with or without 0.3 T magnets. EL-4 cells were used as target cells, which were pulsed with 10 nM OVA257–264 for 30 min. After EL-4 cells and CTLs were each washed three times with PBS, they (1 × 105 for both cell types) were mixed in killing medium  at respective ratios of 1.25:1, 2.5:1 and 5:1. After 6 h, the cytotoxic efficiency was measured using a CytoTox 96 Non-Radioactive Cytotoxicity kit (Promega) which quantified the release of endogenous lactate dehydrogenase (LDH) from EL-4 cells.A CD828. CD8+ CTL cells treated with or without 0.3 T magnets were induced from OT-I mice as previously mentioned. Then, CTLs (3 × 106) were intravenously injected into recipient C57BL/6 mice. After 4 h, CFSElow (1 μM)-labeled splenocytes were pulsed with 10 nM OVA257–264 peptide, mixed at a 1:1 ratio with unpulsed CFSEhigh (10 μM)-labeled splenocytes (5 × 106) and intravenously injected into recipient mice. The OVA257–264-pulsed splenocytes (CFSElow) and nonpulsed splenocytes (CFSEhigh) were used as target cells. Non-pulsed splenocytes were not lysed and used as internal controls. After 6 h, splenocytes from the recipient mice were analyzed by flow cytometry to detect CFSE expression, and cytotoxicity was calculated as [1 − (% CFSElow)/(% CFSEhigh)] × 100%.The in vivo killing assay was performed as previously described with some modifications27. B16F10-OVA cells (2 × 105) were subcutaneously injected into 8- to 10-week old C57BL/6 mice. On day 12, melanoma-bearing mice with similar tumor sizes were randomly divided into three groups (n = 7–10) and intravenously injected with PBS, CTLs (1.5 × 106) generated from OT-I mice treated with 0.3 T magnets or control CTLs (1.5 × 106) treated without magnets. For combined therapy of CTLs and anti-PD-1 antibody, another three groups were formed: anti-PD-1 antibody, CTLs and anti-PD-1 antibody in the presence of 0.3 T magnets, and CTLs and anti-PD-1 antibody in the absence of the magnets. Anti-PD-1 antibody  was intraperitoneally injected every 3 days. Starting on day 12, the tumor size was measured every 3 days, and the animal survival rate was recorded daily. Tumor size was calculated as length × width. Mice with tumor sizes larger than 20 mm on the longest axis were euthanized for ethical purposes.Adoptive T cell transfer was performed as previously described2/2, where A is the sagittal length (mm) and B is the cross length (mm). Mammary tumors from sacrificed mice were dissected, fixed in 4% (v/v) buffered neutral formalin and embedded in paraffin. Five-micron tissue sections were stained with hematoxylin and eosin. All images were observed under an Olympus BX51 microscope and acquired with a DP71 camera (Olympus).PyMT mice (FYB background) were treated with magnetic plates or control plates since 3 weeks old and examined by visual inspection and palpation every 2–3 days to monitor tumor onset. The tumor size was measured weekly with digital Vernier calipers, and the tumor volume was estimated using the formula A × B42. RNA was extracted, purified and checked for integrity using an Agilent Bioanalyzer 2100 (Agilent Technologies). Libraries were generated for sequencing using a SMARTer Stranded Total RNA-Seq Kit-Pico Input Mammalian (Illumina). Libraries were sequenced using an Illumina HiSeq X Ten sequencer. Only genes with a fold change ≥ 2 and a p value ≤ 0.05 were defined as differentially expressed. Gene Ontology biological processes that were enriched based on the differentially regulated genes were identified by DAVID 43.RNA-Seq, library generation and bioinformatics analysis were performed as previously describedNdufc1 (NM_025523.1) ;Atp6v0b (NM_033617.3) ;Idh3b (NM_130884.4) ;Ndufs6 (NM_010888.2) ;Uqcrc2 (NM_025899.2) ;Uqcrb (NM_026219.2) ;Ndufa13 (NM_001382215.1) ;Atp5k (NM_007507.3) ;Uqcrfs1 (NM_025710.2) ;Ndufa10 (XM_030242321.1) ;Gzmb (NM_013542.3) ;Tnf (NM_001001495) ;Ifng (NM_008338.4) ;Isca1 (NM_026921.4) ;Isca2 (NM_028863.1) ;Cry1 (NM_007771.3) ;Cry2 (NM_009963.4) ; and.Beta-actin (XM_030254057.1) .Total RNA from cell samples was extracted with TRIzol (Invitrogen), reverse-transcribed using a SuperScript III First-Strand kit (Invitrogen) and subjected to quantitative reverse transcription PCR (qRT-PCR). The gene-specific primers were designed based on mRNA sequences retrieved from NCBI. The NCBI reference sequence numbers and primer sequences (5′–3′) of the genes are provided as follows:+ T cells from C57BL/6 mice were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 24 h, incubated in media containing retrovirus supernatants and centrifuged at 1,500 g for 2 h at 32 °C. Retroviral supernatants were then replaced by fresh culture medium, and cells were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 72 h in the presence or absence of 0.3 T magnets. GFP-positive cells were sorted and subjected to real-time reverse transcriptase-PCR to test the knockdown efficiency. In order to rule out an off-target effect, we designed at least three shRNAs per gene and only the shRNA having predominant knockdown efficiency was finally used. In addition, the cell samples transfected with empty vector was used as negative controls44.Uqcrb-target shRNA: 5′-TGCTGTTGACAGTGAGCGCAAGAAGTGATCTTTTAGTTAATAGTGAAGCCACAGATGTATTAACTAAAAGATCACTTCTTTTGCCTACTGCCTCGGA-3′. Mouse Ndufs6-target shRNA: 5′-TGCTGTTGACAGTGAGCGCGCCATTGATTTGATAGCACAATAGTGAAGCCACAGATGTATTGTGCTATCAAATCAATGGCATGCCTACTGCCTCGGA-3′.Mouse Isca1-target shRNA: 5′-TGCTGTTGACAGTGAGCGATGCCTCGTGGGTGAAAATAAATAGTGAAGCCACAGATGTATTTATTTTCACCCACGAGGCAGTGCCTACTGCCTCGGA-3′.Mouse Cry1-target shRNA:Mouse 5′-TGCTGTTGACAGTGAGCGCCCGCCTCTTTATTTACATCTATAGTGAAGCCACAGATGTATAGATGTAAATAAAGAGGCGGATGCCTACTGCCTCGGA-3′.Cry2-target shRNA:Mouse 5′-TGCTGTTGACAGTGAGCGCCAGTTTGTTTGTGAATATTTATAGTGAAGCCACAGATGTATAAATATTCACAAACAAACTGTTGCCTACTGCCTCGGA-3′.For the construction of knockdown T cells, Plat-E cells were transfected with MLP shRNA plasmids, and retrovirus supernatants were harvested 48 h after transfection. Purified CD8+ T cells (105) was detected using an ATP Assay Kit (Beyotime) following the manufacturer’s recommendations. The luminescence of each sample was measured using a GLOMAX 20/20 Luminometer.The intracellular ATP concentration in CD8+ T cells under basal conditions and in response to 1 μM oligomycin, 1 μM fluorocarbonyl cyanide phenylhydrazone (FCCP), 1 μM rotenone and 1 μM antimycin, 10 mM glucose and 100 mM 2-Deoxy-D-glucose (2-DG). The link between OCRs and mitochondrial ATP production (ATP-linked OCR) was determined by subtracting the OCR of CD8+ cells reached after treatment with oligomycin from the OCR obtained at baseline. All extracellular flux analyses were performed using an XF-24 Extracellular Flux Analyzer (Seahorse Bioscience) as recommended by the manufacturer.Using the Seahorse MitoStress Test Kit, we analyzed the OCRs and ECARs of CD8t-test was used, unless otherwise indicated, to determine statistical significance. Data are representative of or combined from at least three independent experiments. For all experiments: *p < 0.05; **p < 0.001; ***p < 0.0001, ****p < 0.0001.Statistical analyses were performed with GraphPad Prism 6. Data are expressed as the mean ± SEM, and a two-tailed paired or unpaired Student’s Supplementary Information.


Three {[(isoxazol-3-yl)imino]methyl}phenols were synthesized and structurally characterized. All three structures contain an intramolecular O—H⋯N hydrogen bond and none were found to be strongly thermochromic. E)-2-{[(isoxazol-3-yl)imino]methyl}phenol, C10H8N2O2, (E)-2-{[(5-methylisoxazol-3-yl)imino]methyl}phenol, C11H10N2O2, and (E)-2,4-di-tert-butyl-6-{[(isoxazol-3-yl)imino]methyl}phenol, C18H24N2O2. All three structures contain an intramolecular O—H⋯N hydrogen bond, alongside weaker intermolecular C—H⋯N and C—H⋯O contacts. The C—O(H) and imine C=N bond lengths were consistent with structures existing in the enol rather than the keto form. Despite having dihedral angles <25°, none of the compounds were observed to be strongly thermochromic, unlike their anil counterparts; however, all three compounds showed a visible colour change upon irradiation with UV light.The synthesis and structures of three isoxazole-containing Schiff bases are reported, namely, ( Schiff bases of salicylaldehyde derivatives with aniline derivatives, having been first found to exhibit both thermo- and photochromism in the solid state -2-methoxy-6-{[(5-methylisoxazol-3-yl)imino]methyl}phenol -3,5-di-tert-butylsalicylaldimine -2-{[(isoxazol-3-yl)imino]methyl}phenol, 1, (E)-2-{[(5-methylisoxazol-3-yl)imino]methyl}phenol, 2, and (E)-2,4-di-tert-butyl-6-{[(isoxazol-3-yl)imino]methyl}phenol, 3  derivatives with isoxazole derivatives have not been widely characterized structurally, with a search of the Cambridge Structural Database  and aniline (0.0025 mol) were each dissolved in ethanol (25 ml). The resulting solutions were combined and refluxed with stirring for 6–8 h. Any precipitate was filtered off, rinsed with ethanol and left to dry. The (remaining) solution was then rotary evaporated until (further) precipitate formed. Recrystallization was carried out from hexane–dichloromethane for supporting information.Elemental C, H and N content analysis was carried out using the Durham University Analytical service on an Exeter Analytical E-440 Elemental Analyzer. Mass spectrometry in positive electrospray (ES+) mode was performed by the Durham University Mass Spectrometry service on a Waters TQD with an Acquity solvent system. Full details are available in the Compounds 1 and 2 crystallized in noncentrosymmetric space groups; however, the Flack parameters obtained were not meaningful as the data were collected with molybdenum radiation and there are no heavy atoms to facilitate anomalous dispersion. In 3, which contained two independent molecules in the asymmetric unit, one of the tert-butyl groups was disordered; the sum of the occupancies of the two parts was set to equal 1 and subsequently fixed at the refined values. The interplanar dihedral angle was calculated by measuring the angle between planes computed through the five or six non-H atoms of the two rings. See Table 1All H atoms, apart from the hydroxy H atom involved in intramolecular hydrogen bonding with the imine N atom, were positioned geometrically and refined using a riding model. The H atoms involved in the intramolecular hydrogen bonding were located in a Fourier difference map wherever feasible. 1–3 all consist of the same basic backbone with a hydroxy-substituted arene group joined to an isoxazole ring via an imine (C=N) group  Å in 3], while the C1—O1 bond lengths [ranging from 1.350 (2) Å in 1 to 1.3655 (18) Å in 3] are consistent with a single bond. Indeed, the hydroxy H atom was located in a Fourier difference map in the vicinity of the O atom, supporting the fact that the structures are all in the more commonly observed enol form rather than the keto form. All three structures contain an intramolecular O1—H1⋯N1 hydrogen bond with similar parameters, e.g. the O1⋯N1 distances range from 2.6062 (17) to 2.632 (2) Å  Å]  Å], creating a one-dimensional stack approximately up the [101] direction. All the stacks in the ac plane are in the same direction; however, moving in the b-axis direction by one molecule, the stacks in the ac plane are in different directions due to the presence of the 21 screw axes and glide planes. The structure also contains: (i) C—H⋯N and C—H⋯O interactions involving the N and O atoms of isoxazole; (ii) C—H⋯O interactions involving the O atom of the OH group. These interactions link the central molecule to four others, two on each side of the molecule, creating a three-dimensional network. An illustration of the overall packing is shown in Fig. 3The structure of 3, the two independent molecules show slightly different intermolecular interactions: (i) C—H⋯N  and a C—H⋯O interaction involving the N and O atoms of isoxazole; (ii) C—H⋯O interactions involving the O atom of the OH group. This creates a three-dimensional packing network ° for 2 and 6.53 (10)/14.27 (8)° (two molecules) for 3; however, none of the compounds were observed to be strongly thermochromic by eye when cooled to ∼80 K. In the case of 2 and 3, this is perhaps not a major surprise as they are yellow at room temperature and, while they did become paler in colour at lower temperatures, the strongly thermochromic anil compounds are typically a red/orange colour at room temperature and change to yellow upon cooling. However, 1, which is orange at room temperature, remained an orange colour at ∼80 K also. All three compounds did show evidence of photochromism with a colour change, from orange to red for 1 and from yellow to orange for 2 and 3, upon irradiation with UV light.The chromic behaviour of compounds E)-2-{[(isoxazol-3-yl)imino]methyl}phenol, 1, (E)-2-{[(5-methylisoxazol-3-yl)imino]methyl}phenol, 2, and (E)-2,4-di-tert-butyl-6-{[(isoxazol-3-yl)imino]methyl}phenol, 3, are reported. The three structures all exist in the enol form and display an intramolecular O—H⋯N hydrogen bond. All three structures contain intermolecular C—H⋯N and C—H⋯O contacts. In the structures of 1 and 2, π–π-type contacts were identified between the C=N group and the phenol ring. All three compounds had dihedral angles of <25°; however, none of the compounds were observed to be strongly thermochromic and even 1, which was orange at room temperature, did not show a significant colour change upon cooling. This is in contrast to the anils where orange compounds with a dihedral angle of <25° are normally strongly thermochromic. All three title compounds did show evidence of photochromism upon irradiation with UV light.The structures of three Schiff bases of salicylaldehyde derivatives with isoxazole derivatives, namely,  1, 2, 3, global. DOI: 10.1107/S2053229620010530/wv30011sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2053229620010530/wv30012sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S2053229620010530/wv30013sup4.hklStructure factors: contains datablock(s) 3. DOI: 10.1107/S2053229620010530/wv3001sup5.pdfCharacterization data for compounds 1-3. DOI: 2020495, 2020494, 2020493CCDC references:


Scientific Reports10.1038/s41598-018-19188-2, published online 22 January 2018Correction to:This Article contains errors.The legend of Figure 4 is incorrect:2O spectroscopy at 0.013 mbar over 65 cm−1. Green line: P(40), 37435005.26(10) MHz; blue line: P(18), 38052238.584(63) MHz; red line: R(31), 39268217.812(78) MHz.”“Nshould read:2O spectroscopy at 0.013 mbar over 65 cm−1. P(40), 37435005.58(10) MHz; blue line: P(18), 38052238.986(63) MHz; red line: R(31), 39268218.298(78) MHz”“NAs a result, there is an error in the Results and Discussion section where,“The resulting line centre-frequency of the P(18) line is 38052238584(63) kHz; the HITRAN value for the line-centre frequency is only 2.7 MHz above our determination while the HITRAN value has relatively large nominal uncertainty range of 3–30 MHz”should read:“The resulting line centre-frequency of the P(18) line is 38052238986(63) kHz; the HITRAN value for the line-centre frequency is only 2.7 MHz above our determination while the HITRAN value has relatively large nominal uncertainty range of 3–30 MHz.”