The X-ray diffraction (XRD) pattern of all the nanomaterials showed a C-V2NO single-phase cubic structure. The C-V2NO obtained at 700 °C had a low surface area (91.6 m2 g−1), a moderate degree of graphitization, and a broader pore size distribution. The C-V2NO obtained at 800 °C displayed an interconnected network with higher surface area (121.6 m2 g−1) and a narrower pore size distribution. In contrast, at 900 °C, the C-V2NO displayed a disintegrated network and a decrease in the surface area (113 m2 g−1). All the synthesized C-V2NO yielded mesoporous oxynitride nanostructures which were evaluated in three-electrode configuration using 6 M KOH aqueous electrolyte as a function of temperature. The C-V2NO@800 °C electrode gave the highest electrochemical performance as compared to its counterparts due to its superior properties. These results indicate that the nitridation temperature not only influences the morphology, structure and surface area of the C-V2NO but also their electrochemical performance. Additionally, a symmetric device fabricated from the C-V2NO@800 °C displayed specific energy and power of 38 W h kg−1 and 764 W kg−1, respectively, at 1 A g−1 in a wide operating voltage of 1.8 V. In terms of stability, it achieved 84.7% as capacity retention up to 10,000 cycles which was confirmed through the floating/aging measurement for up to 100 h at 10 A g−1. This symmetric capacitor is promising for practical applications due to the rapid and easy preparation of the carbon-vanadium oxynitride materials.In this work, porous carbon-vanadium oxynitride (C-V The growing demand for conversion of renewable energy systems and a foreseeable shortage of fossil fuels for usable energy is a major challenge for energy storage. However, many high-energy storage systems have also been developed to meet the generated energy efficiently for productive use and cost-effectiveness, such as batteries and supercapacitors ,2. SuperTypically, ultracapacitors can be classified into two types based on their charge storage mechanism: electric double-layer capacitors (EDLCs) and Faradaic capacitors ,7. EDLCs2−) atoms in the metal octahedral position were completely replaced by nitrogen (N3−) atoms, while for the MONs  atom, M = Ti , V 18]))2−) atom, M = Ti .5+, V4+, V3+, V2+) leading to an excellent specific capacity, and the presence of some vanadium oxides. VOxNy still suffers from poor rate performance, low specific surface area and aging effects [2 or V2O5 resulting in the generation of aggregated VOxNy particles owing to the low melting point of the vanadium oxides [Among the greatest recent achievements, vanadium oxynitride materials has been considered to be an excellent candidate due to their superior outstanding electrochemical properties due to multiple oxidation states  is less toxic, less corrosive and safer compared to the chloride-based precursors and thus, a suitable alternative for the preparation of VOxNy materials. Moreover, a recent approach on the use of organic precursors in the growth of oxynitride materials has resulted in a controlled particle formation, generation of a crystalline structure, as well as the ability to tune the specific surface area of the final product [1.02O0.1 material by calcining a mixture of melamine and V2O5 xerogel in a furnace at 800 °C under N2 atmosphere. They obtained a carbon content of 52.6% and a nitrogen content of 22.6% after melamine decomposition [xNy and, as a result, improve their electrochemical capacitance [Although various researchers have reported on the use of vanadium chloride and vanadium oxychloride precursors to obtain good crystalline VOructures ,21, a coructures . Ammoniu product . In this product  studied position . The preacitance  demonstracitance . To the 2NO nanostructures. At 700 °C, a low surface area and less crystalline C-V2NO nanostructure was obtained. In contrast, at 800 and 900 °C, the C-V2NO nanostructures showed higher specific surface area and smaller pore size distribution. The effect of the different nitridation temperature was associated with the electrochemical performances of the C-V2NO electrodes evaluated in 6 M KOH aqueous electrolyte. To confirm the electrochemical properties of the material, a symmetric supercapacitor (C-V2NO//C-V2NO) was assembled using a C-V2NO@800 °C in both positive and negative potential windows. In a wide voltage of 1.8 V, the symmetric device exhibited a specific energy and specific power of 38 W h kg−1 and 764 W kg−1 at 1 A g−1, respectively. The (C-V2NO//C-V2NO) showed 84.7% capacity retention at 10,000th cycles and excellent voltage holding stability up to 100 h at a high specific current.This study presents an in-depth understanding of how nitridation temperature influences the physical and electrochemical properties of C-V2NO nanomaterials at a nitridation temperature of 700 °C. The C-V2NO nanomaterials were synthesized by thermal decomposition method using ammonium metavanadate  and melamine  with a mass loading ratio of 1:10. The preparation details of the C-V2NO was reported in our previous study by Ndiaye et al. [4VO3 and C3H6N6 powders were mixed in an agate mortar with a few drops of ethanol  to make a homogeneous slurry. This was inserted into a quartz tube furnace and heated to 700 °C at a heating rate of 18 °C min−1 before being kept at that temperature for 2 h under a stream of nitrogen (N2) gas flow. After cooling down to room temperature, the final product was obtained and labelled as C-V2NO@700 °C. The same method has been used to prepare C-V2NO nanomaterials at 800 °C and 900 °C.e et al. . Briefly2NO nanomaterials was studied using a cobalt X-ray source at 50 mA and 35 kV by a XPERT-PRO diffractometer  with θ/2θ geometry in the range from 10–90°. Raman spectroscopy analysis of the materials were operated using a WITec confocal Raman microscope  . A field-emission scanning electron microscope of a Zeiss Ultra plus 55 field (FE-SEM)  was used to investigate the morphology of the materials and the elemental mapping operated at a voltage of 2.0 kV. Transmission electron microscopy (TEM) analysis was carried out at 200 kV on a JEOL JEM-2100F microscope .X-ray diffraction (XRD) analysis of as-prepared C-V2NO nanomaterials were evaluated with nitrogen adsorption-desorption isotherms at −196 °C using a Micromeritics ASAP 2020 . The specific surface area values of the materials were obtained by the Brunauer–Emmett–Teller (BET) analysis using the adsorption branch in the relative pressure range (P/P0) of 0.01–1.0.X-ray photoelectron spectroscopy  was used to study the composition of the materials with a monochromatic Al-Kα radiation. The porosimetry tests of the C-V2NO electrodes at room temperature. The C-V2NO electrodes were investigated by mixing weighed amounts of C-V2NO active material, carbon black additive and polyvinylidene difluoride (PVDF) binder in a mass ratio of 80:10:10 with N-methylpyrrolidone (NMP) as a solvent. The slurry was applied on a clean nickel foam (NiF) with a 3D scaffold template as a current collector. The electrodes were kept at 60 °C in an electric oven overnight to ensure a complete evaporate of the NMP. Ag/AgCl (3 M KCl saturated) was used as reference electrode, a glassy carbon plate as counter electrode in a 6 M KOH aqueous electrolyte. The C-V2NO electrodes were used as working electrodes to evaluate the electrochemical performance.A Bio-Logic VMP-300  potentiostat were used to evaluate the electrochemical measurements (three- and two-electrode configurations) of the C-VS, mA h g−1) of the electrodes’ materials was calculated using Equation (1) [dI is defined as the specific current measured in A g−1 and Dt is the time in second (s) for a complete discharge cycle.The measurements of the cyclic voltammetry (CV) and galvanostatic charge-discharge (GCD) were studied to determine the behaviour, the stable working potential and to calculate the specific capacity and the stability test of the electrodes, respectively. The specific capacity (Qtion (1) .(1)Q=IddE,W h kg−1) and the specific power,  of the C-V2NO//C-V2NO device were calculated using Equations (2) and (3) at different specific currents [dI (A g−1) represents the specific current, Dt (s) is called discharge time from the GCD curve, and V (V) is the voltage of the symmetric capacitor.The specific energy (currents .(2)Ed card #43182 [2NO was found to increase with nitridation temperature indicating an increase in crystallinity. Indeed, the peaks of C-V2NO@800 °C and C-V2NO@900 °C were narrower and much pronounced than the ones for C-V2NO@700 °C. However, the diffraction peak of carbon was not observed in XRD analysis results of the carbon-vanadium oxynitride nanostructures synthesized at 700, 800 and 900 °C, respectively, are presented in d #43182 . The pea2NO nanostructures at different nitridation temperatures. The D peak position was observed at (1346–1373 cm−1) as indicated in −1) is related to the vibration modes of the graphitic sp2 carbon present [2NO nanomaterial. The D’ peak is ascribed to the lattice vibration mode of the G peak at the surface of the carbon structure [The existence of amorphous carbon in the nanomaterials was nonetheless further confirmed from the Raman spectra as shown in  present ,30. A smtructure ,32. A sitructure .D/IG ratio of the C-V2NO@800 °C recorded in D/IG ratio) in the C-V2NO@800 °C can contribute to high electrochemical capacity [2NO nanomaterials synthesized at 700, 800 and 900 °C also displayed Raman bands at wavenumbers of 142, 145, 192, 285, 479, 520.7, 693, 822, 873, and 995 cm−1 as seen in 2 and V2O5) stretching mode [The high Icapacity ,35. The ing mode .2NO nanomaterials synthesized at different temperatures is presented in 2NO nanomaterials at different temperatures exhibited an agglomeration of cubic structures which showed a web-like morphology on the surface with irregular cavities  analysis was carried out to determine the elements present in the materials. The elemental mapping displayed the distinct element distribution in the C-V2NO nanostructure was obtained from transmission electron microscopy (TEM) studies. 2NO@800 °C nanomaterials was evaluated and presented in A more detailed analysis of the C-V2 adsorption-desorption measurement was carried out on C-V2NO nanomaterials at 700, 800 and 900 °C, as shown in 2 isotherm of the C-V2NO nanomaterials in 2 g−1, 121.6 ± 0.83 m2 g−1 and 113 ± 0.74 m2 g−1 at 700, 800 and 900 °C, respectively. The synthesis of these mesoporous interconnected web-like structures with controlled growth temperature yielded higher value of the specific surface area as compared to those reported in the literature namely, 28.8 m2 g−1 for VOxNy, 40.8 m2 g−1 for VOxNy-C [2 g−1 for TiOxNy [To investigate the porosity properties of the nanomaterials, N VOxNy-C  and 60 mr TiOxNy , respect2NO material. It shows clearly that the pore diameter of the C-V2NO nanomaterial decrease with increasing nitridation temperature. The C-V2NO@700 °C shows a broad pore size distribution whereas a narrow pore size distribution was observed for the C-V2NO nanomaterials at 800 and 900 °C. As seen in 2NO@800 °C exhibited a higher specific surface area and a higher pore volume than the C-V2NO@700 °C and C-V2NO@900 °C which could be due to the highest ratio of disordered carbon relative to the graphitic carbon with a small pore diameter of 2 nm. The pore diameter value of the C-V2NO@800 °C (2 nm) is smaller than those reported for most reports on mesoporous oxynitride-based materials such as mesoporous bimetallic molybdenum tungsten oxynitride (4 nm) [2 (20 nm) [2NO@800 °C could greatly increase the mass transport of electrolytes ions within the electrodes for fast redox reactions and thus further enhance the electrochemical performance.e (4 nm) , zirconi (20 nm) , titaniu (20 nm) . This me2, –C–N and nitrogen groups which form the matrix ring (N–C matrix) at higher temperatures [4VO3 decomposes at (>300 °C) to yield vanadium oxides, NH3 gas and H2O [3 gas can further reduce the vanadium oxides in the presence of –CH2 and N–C matrix from melamine decomposition leading to the formation of vanadium oxynitride material at higher temperatures (500–1500 °C) [3, favoured the formation of the carbon-vanadium oxynitride at high nitridation temperatures.The formation of mesoporous carbon-vanadium oxynitride can be postulated to result from the reaction of vanadium metavanadate and melamine at higher temperatures. Typically, at high temperatures (>220 °C), the melamine decomposes to give –CHeratures  and the  and H2O ,44. The 1500 °C) . The pre2NO were formed and connected in a web network with a moderate degree of graphitized carbon.At 700 °C, small nanoparticles of C-V2NO surface with the electrolyte. Consequently, the carbon network became more defective resulting in a lower degree of graphitization. At 900 °C, however, the highly mesoporous network was disintegrated resulting in a decreased specific surface area and a moderately graphitized carbon network. Thus, nitridation temperature influenced the morphology, structure and surface area of the C-V2NO.When the temperature was further increased to 800 °C, the particles are strongly interconnected to form a more mesoporous network (higher surface area) that can favour a better interaction of the C-V2NO prepared at 800 °C portrayed good crystallinity, a higher ratio of disorder with a well-interconnected porous structure. More specifically, it exhibited a high specific surface area and yielded an efficient nanometer-sized pores (2 nm) and therefore, presented an optimal nitridation process that could be beneficial for enhanced electrochemical performance. Thus, the XPS analysis of the C-V2NO@800 °C was performed to determine the active species introduced into the material. 1s V2p, O1s and N1s which confirms the configurations of carbon, nitrogen, oxygen and all the different vanadium ions  [For all materials analysed, C-Vand V5+) ,54,55,562NO nanomaterials in both positive (0 to 0.40 V) and negative (−1.20 to 0 V) potential window ranges at a 50 mV s−1 scan rate. As observed in these figures, the CV plots of C-V2NO@800 °C exhibits a higher current response compared to the other C-V2NO samples prepared at 700 and 900 °C in both operating potential ranges. All the CV curves of the electrodes present Faradaic behavior in the positive potential window and pseudocapacitive behavior in the negative potential window. As seen in 2NO@800 °C electrode presents the highest specific capacity value as a function of the nitridation temperature in the positive and in the negative potential windows.The electrochemical measurements of all the electrode materials were originally performed in a half-cell (three electrode) configuration using 6 M KOH aqueous electrolyte as seen in 2NO electrodes. More specifically, the C-V2NO@800 °C electrode shows smallest ESR, a shorter diffusion length and is closer to the vertical line (Z”-axis) than the other electrode materials. This low ESR value for 800 °C can be related to a good ion diffusion at the electrode–electrolyte interface and fast charge transport between the electrode and the current collector which are greatly influenced by the surface chemistry of the C-V2NO@800 °C electrode.2NO@800 °C depicted a superior electrochemical response based on the result in −1 as shown in the supplementary information  and negative (−1.2 to 0 V) potential window ranges at different scan rates ranging from 5 to 100 mV s−1. The CV curve depicts a Faradaic electrode material in the positive potential window as seen in −1 which correspond to the anodic peak (−0.12 V) and cathodic peak (−0.35 V). These Faradaic and pseudocapacitive processes in the positive and negative potential windows are due to the presence of the vanadium oxide in the material which indicates a reversible redox reaction . Based o2NO@ 800 °C calculated in the positive and the negative potential window at different specific currents ranging from 1 to 10 A g−1. The specific capacities of the C-V2NO@800 °C were found to be 55 and 67 mA h g−1 in the positive and in the negative potential window, respectively, at a specific current of 1 A g−1.2NO@800 °C electrode extensively, a symmetric cell was fabricated using the materials prepared at 800 °C as both positive and negative electrodes (C-V2NO//C-V2NO) in the same electrolyte (6 M KOH). 2NO@800 °C measured in stable working potential windows of −1.2 to 0 V and 0 to 0.4 V, respectively, at a scan rate of 50 mV s−1, and an estimation of the working potential window of 1.6 V could be expected from the symmetric device.To further demonstrate the electrochemical performance of the C-V−1. The cyclic voltammogram curve at 1.8 V shows the highest current response and good contribution of the two behaviours of C-V2NO@800 °C in both positive and negative voltages compared to other (1.6 and 1.7 V). Moreover, the current leap at large voltage of 1.8 V was not observed, implying the stability of the symmetric device in this voltage  resistance, and R2 and Q2 represent the rate of resistance and the electrochemical Faradaic activity, respectively. The equivalent series resistance is associated with the ohmic resistance of the electrode. In the high frequency, the R1 in series with the constant phase element Q2 and is placed in parallel with the R2. In the low-frequency, a close vertical line has been shown which is associated with an ideal supercapacitor giving a mass capacitance, Q3. The Nyquist plot of the symmetric device presents a slight deviation from the Y-axis which is mainly associated with the leakage resistance R3 arising from the Faradaic charge transfer process. The Q3 is in parallel to the R3 which is in series with the Warburg impedance characteristic element (W) [ment (W) ,60.−1 of the symmetric device was recorded at 1 A g−1. This was retained at 18.7 mA h g−1 for a specific current of 10 A g−1. −1. The specific energy of the C-V2NO//C-V2NO was 38 W h kg−1 with a related specific power of 764 W kg−1 at a specific current of 1 A g−1. At 10 A g−1 the specific energy was 12.4 W h kg−1 with an equivalent specific power of 6.6 kW kg−1. These values are superior to those reported for other symmetric oxy/nitride-based materials as shown in 2/TiOx1−Nx nanomaterials by depositing MnO2 on TiO0.54N0.46 nanotubes. The TiOx1−Nx was prepared by using a CVD  system in an ammonia atmosphere at high temperature. The symmetric configuration delivered an specific energy value of 9.8 W h kg−1 and a specific power value of 620 W kg−1 at 100 A g−1 using 1 M KCl electrolyte [−1 and deliver a specific power of 160.5 W kg−1 at a specific current of 1 A g−1 in PVA/H2SO4 electrolyte [ctrolyte . Recentl2NO//C-V2NO cell observed can be attributed to the excellent electrochemical performance of the C-V2NO@800 °C working in a stable voltage of 1.8 V.In this work, the high specific energy and power value of the C-V2NO//C-V2NO, constant cycling (charging–discharging) and floating time (voltage holding) tests were performed to study device degradation with varying parameters. −1.To evaluate the device stability of the C-V−1 by monitoring the specific capacity after every 10 h interval for up to 100 h and the result is shown in −1 before reaching a plateau at a value of 32 W h kg−1, corresponding to an increase of 27% from the original value of 23 W h kg−1. This increase in specific capacity and specific energy can be associated to the increase in accessibility of the ions to initially inaccessible redox sites which could increase the wettability and a large diffusion of ions between the electrolyte/electrode during the long floating time (100 h) [The cycling stability presented a good capacity retention of 84.7% up to 10,000 cycles, suggesting a good electrochemical stability of the symmetric cell. The floating test was evaluated after 10,000 stability test at 10 A g (100 h) . The cyc2NO) nanostructure was obtained at different nitridation temperatures of 700, 800 and 900 °C using a thermal decomposition method. All the synthesized nanomaterials exhibited single phase C-V2NO cubic structures. The BET analysis of the C-V2NO nanomaterials showed a good specific surface area (SSA) (from 91.6 to 121.6 m2 g−1) with a pore diameter ranging from −2 to 8 nm. The nitridation temperature played a crucial role in modulating the C-V2NO crystallinity, surface area, and elemental composition. The C-V2NO@700 °C nanostructure displayed a low surface area and a moderate degree of graphitization. The C-V2NO@800 °C nanomaterials showed a higher ratio of disorder with a well interconnected cavities structure and a narrower pore size distribution. In contrast, C-V2NO@900 °C gave a disintegrated network with a moderate degree of graphitization and a decrease in surface area compared to the C-V2NO@800 °C. Therefore, a nitridation temperature of 800 °C was found to be the optimal temperature for the generation of a suitable C-V2NO nanostructure with the highest electrochemical performance in both the positive and negative potential windows. A symmetric capacitor was fabricated using C-V2NO@800 °C revealing a specific energy value of 38 W h kg−1 and a specific power value of 764 W kg−1 at 1 A g−1 in a voltage of 1.8 V. The C-V2NO//C-V2NO exhibited a good cycling stability with 84.7% capacity retention up to 10,000 cycles at 10 A g−1. This excellent stability performance of the device was also confirmed via floating/aging test for up to 100 h showing a further improved device electrochemical performance.In this study, a porous carbon-vanadium oxynitride (C-V


A small fraction (ca. 25%) of the population that reaches the S1(nπ*) state repopulates the ground state. The T1(ππ*) state decays to the ground state in 1.4 ± 0.2 μs under N2-purged conditions, using a 0.2 mM concentration of 6-thioguanine, or it can sensitize singlet oxygen in 0.21 ± 0.02 and 0.23 ± 0.02 yields in air- and O2-saturated solution, respectively. This demonstrates the efficacy of 6-thioguanine to act as a Type II photosensitizer. N9-glycosylation increases the rate of intersystem crossing from the singlet to triplet manifold, as well as from the T1(ππ*) state to the ground state, which lead to a ca. 40% decrease in the singlet oxygen yield under air-saturated conditions. Enhanced vibronic coupling between the singlet and triplet manifolds due to a higher density of vibrational states is proposed to be responsible for the observed increase in the rates of intersystem crossing in 6-thioguanine upon N9-glycosylation.6-Thioguanine, an immunosuppressant and anticancer prodrug, has been shown to induce DNA damage and cell death following exposure to UVA radiation. Its metabolite, 6-thioguanosine, plays a major role in the prodrug’s overall photoreactivity. However, 6-thioguanine itself has proven to be cytotoxic following UVA irradiation, warranting further investigation into its excited-state dynamics. In this contribution, the excited-state dynamics and photochemical properties of 6-thioguanine are studied in aqueous solution following UVA excitation at 345 nm in order to provide mechanistic insight regarding its photochemical reactivity and to scrutinize whether N9-glycosylation modulates its phototoxicity in solution. The experimental results are complemented with time-dependent density functional calculations that include solvent dielectric effects by means of a reaction-field solvation model. UVA excitation results in the initial population of the S Sulfur-substituted purine nucleobases have long been prescribed to treat inflammatory bowel disease, arthritis, and various cancers ,3,4,5,6.1O2) yield of ca. 25% . In. In29]. perature , the emiompounds ,40, the ompounds ,42. The 2(ππ*) state is expected to be populated overwhelmingly  level of theory. Vertical excitation energy calculations were performed subsequently on the optimized geometry in the corresponding solvent environment. A reaction field was used to model the solvation effects from water. We note, however, that it has been shown recently that explicit solvent effects are needed to most accurately describe the excitation energies in DNA bases and their thio-derivatives ,44,45. Telmingly  due to ielmingly . This is2(ππ*) state to T2(nπ*) state, or from the S1(nπ*) state to T1(ππ*) state, is favorable, as the change in state character is in agreement with the El-Sayed propensity rules  level of theory [2(ππ*), S1(nπ*), T3(ππ*), T2(nπ*), and T1(ππ*) vertical excitation energies of 6tGua are all within 0.1 eV of those computed for 6tGuo. This similarity leads to nearly identical energy gaps between the S2(ππ*) and T3(ππ*), S2(ππ*) and S1(nπ*), S2(ππ*) and T2(nπ*), and the S1(nπ*) and T1(ππ*) states. The TD-DFT calculations performed in this work can be compared directly with those computed previously for the f theory . As depiThe femtosecond transient absorption kinetics of 6tGua were globally fitted to a two-lifetime exponential model b. Simila1 state is significantly quenched in the presence of molecular oxygen (The full excited-state decay of 6tGua was monitored using nanosecond transient absorption spectroscopy (~400 ps instrument response function) in air and in deaerated solution . The timr oxygen , suggest2-saturated aqueous solution  in the transient absorption data of 6tGua corresponds to the rise of the maxima at 375 nm and 520 nm, while the second (τ2) corresponds to the decay and rise of the 520 nm and 375 nm bands, respectively. In addition, the negative signal at ca. 350 nm appears to repopulate slightly during τ1, but this may result from an increase in excited-state absorption on top of the depopulated region. The most negative ground-state depopulation amplitude (−12 mOD) occurs at a 410 fs time delay  and maximum at 375 nm increase by ~2 mOD, suggesting that the apparent ground-state repopulation in 1 to ultrafast population of the triplet manifold in 6tGua, a process that has been reported for 6tGuo and many other sulfur-substituted DNA derivatives [The first lifetime  state to the T2(nπ*) state, or from the S1(nπ*) state to the T1(ππ*) state, should be most favorable, as they both obey the El-Sayed propensity rules [2(ππ*)/S1(nπ*) conical intersection is accessible barrierlessly from the Frank-Condon (FC) region of the S2(ππ*) state potential energy surface (PES), suggesting that internal conversion from the S2(ππ*) state to the S1(nπ*) state occurs rapidly (~10 fs). The triplet manifold is populated subsequently, primarily through the dark S1(nπ*) state, as has be shown for other sulfur-substituted and canonical DNA bases [1 PES exhibits both ππ* and nπ* minima with energies of 3.78 eV and 3.18 eV, respectively, while the T1 state PES has two ππ* minima at 3.00 eV ((3ππ*)min2) and 3.37 eV ((3ππ*)min) [1 state, initially populating the T2 nπ* minimum and the (3ππ*)min2, which are isoenergetic [1 ππ* minimum to the T2(nπ*) state, and from the S1 nπ* minimum to (3ππ*)min, are expected to be minor contributions to the overall ISC dynamics [1 to the combination of internal conversion from the S2 state to the S1 state, ISC from the S1 state to the T2 state (or T1 state), and internal conversion from the T2 state to the T1 state. As discussed above, TD-DFT vertical excitation energies  indicatety rules . These pty rules . Howeverty rules ,58. ThesNA bases ,49,59. Tππ*)min) . Nonadianergetic ,58. Path2) observed in the ultrafast relaxation dynamics of 6tGua is more difficult to assign. During this timeframe, the spectral features are nearly identical to those observed for 6tGuo, in which decay of the 520 nm band coincides with a rise of the 375 nm maximum [2 was assigned to a combination of solvation dynamics in the T1(ππ*) state and internal conversion from the S1 state to the ground state [1, this may be due to a rise in excited-state absorption on top of the ground-state depopulation signal (1 ππ* minimum and the ground-state PES [1 state to the ground state would last for tens of picoseconds [2) obtained from global analysis of the transient absorption data. These calculations support the assignment of τ2 to internal conversion from the S1 state to the ground state, but they cannot explain the rise in magnitude of the transient absorption band centered at 375 nm. For 6tGuo, τ2 was shown to decrease significantly from 80 ± 15 to 32 ± 5 ps in going from PBS to acetonitrile, which is consistent with the assignment of solvent dynamics in the T1(ππ*) state on an equal time scale. Therefore, we suggest that τ2 in 6tGua corresponds to a combination of internal conversion from the S1 state to the ground state and solvent dynamics in the T1(ππ*) state, as suggested previously for 6tGuo [2, and taking into consideration the rise in absorption of the 375 m band, we estimate an upper limit of 25% for internal conversion to the ground sate. We stress that this is simply an estimation, not a quantitative yield.The second lifetime , T2(nπ*), and T1(ππ*) states upon N9-glycosylation, allowing for more effective vibronic coupling between the singlet and triplet manifolds. While the transient absorption spectral evolution of 6tGua and 6tGuo are nearly identical in aqueous solution, they exhibit significantly different excited-state kinetics. For each, τin 6tGuo . An incrsylation , indicat1O2 quantum yield in going from O2- to air-saturated aqueous solutions, while that for 6tGua remains constant within the error. A similar decrease in triplet-state lifetime was observed for 2-thiothymine upon N1-glycosylation in deaerated acetonitrile [1 state and the ground state. At this time, a similar argument can be suggested to explain the enhanced rate of triplet decay for 6tGua upon N9-glycosylation. Further investigation is needed to scrutinize whether this is a general phenomenon observed in other sulfur-substituted nucleobases.The most notable differences between 6tGua and 6tGuo dynamics are observed at the nano- and microsecond timescales . The trionitrile , and it 2-Amino-6-mercaptopurine  was obtained from Sigma Aldrich  and used as received. Solutions of 6tGua were prepared in aqueous phosphate buffer saline (PBS) at pH 7.4. PBS was prepared using 0.480 g of sodium dihydrogen phosphate and 0.354 g of disodium hydrogen phosphate dissolved in 400 mL of ultrapure water and adjusted to pH 7.4 with concentrated sodium hydroxide. Room-temperature absorption and emission spectra were recorded using a Cary Bio 100 spectrophotometer . Samples were prepared in cuvettes with 1 cm optical path length. Ground-state molar extinction coefficients were measured for 6tGua in PBS by making serial dilutions from a prepared stock solution. Steady-state and time-resolved luminescence spectra at 77 K were recorded on a Fluorolog-3 fluorimeter  using 3 mm (inner diameter) quartz tubes inside a quartz liquid nitrogen dewar. The phosphorescence lifetime at 77 K was measured by multichannel scaling on an OB920 spectrometer  in conjunction with a pulsed Xe-lamp.2-saturated conditions. Samples were purged for at least 30 min in septum-top cuvettes prior to data collection. Excitation spectra were recorded at each of the emission maxima under air-saturated conditions. Both the emission and excitation spectra were collected at a scan rate of 20 nm/min with the excitation and emission slit widths set to 5 nm and the photomultiplier tube gain set to 800 V.Room-temperature emission spectra were obtained using a Cary Eclipse spectrofluorimeter . Samples were prepared in cuvettes with 1 cm optical path length. Ground-state molar extinction coefficients were measured for 6tGua in PBS by making serial dilutions from a prepared stock solution. Emission spectra were measured at room temperature using an excitation wavelength of 340 nm under air- and NQuantum-chemical calculations were performed using the Gaussian 09 suite of programs . Ground-TD-DFT calculations have been shown to satisfactorily model the electronic structure of the DNA nucleobases and nucleosides and their thio-derivatives ,68,69,702 crystal to generate a broadband probe pulse (320 nm–710 nm). For each molecule, the OPA was tuned to excitation wavelength of 345 nm. Excitation pulses were set to an intensity of 1 µJ at the sample using a neutral density optical filter in order to minimize cross-phase modulation effects as well as sample degradation [2-saturated solutions. N2-saturated solutions were purged for 30 min prior and blanketed with N2 for the course of the experiment. Degradation of 6tGua was monitored at 340 nm in PBS. Samples were replaced with fresh solutions before the change in absorbance reached 5%. Femtosecond broadband transient absorption spectroscopy was employed to study the excited-state dynamics of 6tGua. The laser and spectrometer setup is described in significant detail in previous publications ,73. In bradation . The setradation ,73. DuriA homemade software  was used for data collection. Prior to analysis, the data was corrected for group velocity dispersion of the probe pulse using the two-photon absorption signal of neat methanol. Approximately 80 kinetic traces were extracted from the multidimensional transient absorption data for global and target analysis with Igor Pro 6.36 . Traces 1O2 produced by 6tGua following UVA excitation [1O2 phosphorescent decay traces were collected at 1270 nm using a modified Fluorolog-3 spectrometer  with a NIR sensitive photomultiplier tube . The decay traces were then stored on a digital oscilloscope . Solutions of 6tGua and the phenalenone standard were prepared in Tris-buffered D2O at an optical density of 0.3 at 355 nm in 1 cm path length quartz cuvettes. The O2-saturated solutions were bubbled with ultrapure oxygen gas for >20 min prior to testing. Degradation of the samples was determined to be less than 3% over the course of the experiments based on their steady-state absorption spectra. The quantum yields were determined in back-to-back luminescence experiments of 6tGua and phenalenone solutions under identical conditions, using the reported yield of 1O2 generated by phenalenone (ΦΔ = 0.98) [Nanosecond time-resolved luminescence spectroscopy was employed to determine the quantum yield of citation ,28,54. B = 0.98) . 1O2 with quantum yields of 0.21 (air) and 0.23 (O2) in PBS, indicating that the nucleobase has the potential to act as Type II photosensitizer before metabolization in the cell cytoplasm. These yields are in good agreement with those reported recently for 6tGuo [The excited-state dynamics of 6tGua have been studied from femtosecond to microsecond time-delays following UVA excitation under physiological conditions. TD-DFT computations and previous high-level quantum-chemical calculations for 6tGua were used to assist in the assignment of the relaxation pathways. We show that 6tGua populates the triplet manifold within ~600 fs followed by decay of the excited-state population from the lowest-energy triplet state to the ground-state in 1.4 and 0.8 μs in deaerated and in air-saturated aqueous solution, respectively. Upon UVA excitation, 6tGua produces or 6tGuo , but appor 6tGuo . They aror 6tGuo ,54.To provide insight into the effects of N9-glycosylation on the excited-state dynamics of 6tGua, we compared our results with those previously reported for 6tGuo under similar conditions. Notably, 6tGuo populates the triplet manifold nearly twice as fast as 6tGua following UVA excitation. The same effect was recently reported for 2tThy, in which N1-glycosylation decreases the magnitude of the ISC lifetime by ~200 fs . Additio


In mammalian neurons, small conductance calcium-activated potassium channels (SK channels) are activated by calcium influx and contribute to the afterhyperpolarization (AHP) that follows action potentials. Three types of SK channel, SK1, SK2 and SK3 are recognized and encoded by separate genes that are widely expressed in overlapping distributions in the mammalian brain. Expression of the rat genes, rSK2 and rSK3 generates functional ion channels that traffic to the membrane as homomeric and heteromeric complexes. However, rSK1 is not trafficked to the plasma membrane, appears not to form functional channels, and the role of rSK1 in neurons is not clear. Here, we show that rSK1 co-assembles with rSK2. rSK1 is not trafficked to the membrane but is retained in a cytoplasmic compartment. When rSK2 is present, heteromeric rSK1-rSK2 channels are also retained in the cytosolic compartment, reducing the total SK channel content on the plasma membrane. Thus, rSK1 appears to act as chaperone for rSK2 channels and expression of rSK1 may control the level of functional SK current in rat neurons. A subset of potassium channels expressed in central neurons are Ca2+ to calmodulin  in hippocampal pyramidal neurons that are generated by the activation of SK2 channels . Biotinylated proteins were captured from the supernatant by the addition of 25 μl Streptavidin Agarose Resin (Pierce) at 4°C for 30 min. The beads were then centrifuged at 10,000 g for 1 min and the resulting supernatant was classified as the cytoplasmic phase (CYTO). The beads were washed a further three times in PBS. This was classed as the membrane fraction (MEMB). All samples were then boiled for 5–10 min in 1× SDS sample buffer containing 100 mM DTT and then fractionated on 4%–12% Bis-Tris gradient gels (Life Technologies) and transferred to polyvinylidene difluoride membrane  at 150 V in 1× MOPS transfer buffer (Life Technologies). Blots were blocked in Tris-buffered saline (TBS) containing 5% skim milk powder, probed with primary antibody  followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG  and detection by SuperSignal West Pico or FEMTO chemiluminescent substrate (Pierce). Blots were scanned using the Odyssey Infrared Imaging System (Li-cor) and densitometry analysis was carried out using Image Studio Lite software.Hippocampal neurons were cooled on ice and were then rinsed three times with phosphate buffered saline (PBS) prior to exposure with EZ-Link Sulfo-NHS-SS-Biotin (Pierce), dissolved in PBS to a concentration of 1.22 mg/ml. The biotinylation reaction was undertaken on ice for 30 min, followed by biotin removal and washing twice with 100 mM glycine in PBS and then once with PBS. Cells were lysed in 200 μl lysis buffer  and left on ice for 30–45 min. The lysates were then centrifuged at 6,500 t-test was used. If the data did not pass the normality test, the Mann-Whitney test was used. Significance was determined at p < 0.05.All data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism (GraphPad Software). All the data were tested for normal distribution using a normality test. If data were normally distributed, a Student’s unpaired Supernatants were precleared for 1 h at 4°C with 75 μl of 50% Sepharose bead slurry (Amersham) and 1.5 μg of species-specific IgG . Protein G sepharose bead slurry and mouse IgG were used for the samples immunoprecipitated with mouse α Myc ; protein A sepharose bead slurry and rabbit IgG for the samples immunoprecipitated with the rabbit α HA  antibody. Samples were then spun at 13,000 rpm for 5 min and the resulting supernatants were incubated overnight at 4°C respectively with mouse α Myc antibody (2 μl/1.5 mg proteins) or rabbit α HA (1:50) antibodies. The following day, the α Myc samples were incubated with 75 μl of 50% protein G sepharose beads and the α HA samples with 75 μl of 50% protein A sepharose beads for a further 3 h at 4°C. Samples were centrifuged at 3,000 rpm for 5 min at 4°C and the recovered pellets were washed three times in RIPA buffer . Samples were eluted with sample buffer containing DTT and denatured by boiling for 5 min. Co-immunoprecipitation samples (IP) were run together with the input (IN) and the supernatant (S) resulting from the immunoprecipitation, transferred as described above and blotted with mouse α Myc and rabbit α HA as described above.HpaI and XhoI. The pll3.7 dsRed vector has the EGFP of pll3.7 vector replaced with dsRed. HEK293T cells were plated at a density of 5 × 105 cells per 35 mm well and were transfected 2 h later using Lipofectamine 2,000 (Life Technologies) as per the manufacturer’s instructions with constructs expressing rat SK1-YFP (gift of G. Moss) and three different plasmids carrying SK1 knock down hairpins  at a ratio of 1:1. Forty-eight hours later, the cells were checked for YFP and dsRed expression, washed with PBS and lysed in 600 μl 1× sample buffer. Thirty microliters of this was run on an SDS-PAGE gel, transferred and western blots carried out using rabbit α GFP  primary antibody as per methods above except that x-ray film was used for detection. Mouse β-actin  was used as a loading control. Out of the three plasmids, rSK1-KD 3.6 was the most successful at reducing SK1-YFP expression  in pll3.7 dsRed with the hSyn promoter using the Not and Nhe restriction sites and the following primers–Synapsin400-Not-F: aaagcggccgcgtgtctagactgcagagggccctSynapsin400-Nhe-R: aaagctagcttctcgactgcgctctcaggA scrambled (SCRAM) control plasmid was also produced using a random combination of the hairpin sequence of the successful knockdown construct. The primers used to produce the SCRAM construct are as follows–SCRAM pLLrSK1_3S: tggcactttaacgccaatcactttcaagagaagtgattggcgttaaagtgccttttttcSCRAM pLLrSK1_3AS: tcgagaaaaaaggcactttaacgccaatcacttctcttgaaagtgattggcgttaaagtgccaThe rat SK2 overexpression plasmid pJPA5.rSK2.Myc3 (gift of J. Adelman) has a triple Myc tag located extracellularly between the S3 and S4 domains. The Myc-tagged SK2 was further cloned into the lentiviral FUGW plasmid (gift of P. Osten) using the AgeI and EcoRI restriction sites and the following primers–F-rSK2: gggaccggtgaaatagccatgagcagctgcaggtacaacgggR-rSK2: ggggaattcgctactctcagatgaagttggThe rat SK1 overexpression plasmid pcDNA3.1-rSK1_HA (gift of G. Moss) has a HA tag located intracellularly between the S2 and S3 domains. The HA-tagged SK1 was further cloned into the lentiviral FUGW plasmid (gift of P. Osten).2 was added together with 3 ml of 2× BBS . This was incubated for 20 min at room temperature before adding the mix dropwise to the cells. The cells were incubated for 3–4 h at 37°C plus 5% CO2 before the removal of the calcium phosphate mix and addition of new media. Viral supernatants were removed 48 h post-transfection, filtered using a 0.45 μm filter unit and then centrifuged for 4 h at 20,000 g over a 20% sucrose cushion. The resulting pellet was resuspended overnight in PBS containing 1% BSA and then ultracentrifuged at 44,000 g for 90 min to concentrate the virus. The virus pellet was resuspended in PBS containing 1% BSA.For lentivirus production, all plasmids  were prepared using the Qiagen Endofree maxiprep kit and lentivirus was prepared via calcium phosphate transfection of 80% confluent triple T175 flasks of HEK293T cells. Cells were grown in DMEM (Life Technologies) plus 10% FBS and 1% penicillin/streptomycin (Life Technologies). Forty microgram of transfer vector together with 20 μg of the other three plasmids were transfected per triple T175 flask. Plasmids were diluted to 0.5 μg/ml in TE buffer and made up to a volume of 3 ml with sterile distilled water. Three-hundred microliter of 2.5 M CaClNeuronal cultures were infected at 4–5 DIV and the virus was left on for 4–12 h. The virus was then removed and conditioned media added to the cultures. Biotinylation experiments and quantitative PCR (qPCR) were undertaken 4–5 days post infection. Knockdown and overexpression lentiviral constructs were also tested for specificity and expression using qPCR. Briefly, RNAs were purified using a RNeasy kit (Qiagen), DNase 1 (Qiagen) treated and reverse transcribed to produce complementary DNA using random hexamers and the SuperScript III First Strand Synthesis System (Life Technologies), as per the manufacturer’s instructions. Standard qPCR was carried out on a Rotorgene RG-3000 Thermocycler using the Platinum SYBR Green qPCR UDG SuperMix (Life Technologies) and 0.2 μM of the following primers:rSK1 Fwd—tca tct cca tta cct tcc tgrSK1 Rev—agc ctg gtg tgt ttg tag atrSK2 Fwd—gtc gct gta ttc ttt agc tct grSK2 Rev—acg ctc ata agt cat ggcrGAPDH Fwd—gag tct act ggc gtc ttc acrGAPDH Rev—cca tcc aca gtc ttc tga gtReactions were performed in either duplicate or triplicate. The PCR program was as follows: 50°C 2 min, 95°C 10 min, 35 cycles −95°C 10 s, 54°C 15 s, 72°C 20 s. Relative expression of rSK1 and rSK2 mRNA was obtained using the comparative Ct method.For the Cosm6 transfections- pJPA5.rSK2.Myc3 and pcDNA3.1-rSK1_HA along with pcDNA3 for the single construct transfections were transfected using the same protocol as described above but using a ratio of 1:2:1.Hippocampal cultures were washed in PBS and fixed in 4% PFA containing 4% sucrose. After three washes in PBS, the cells were first blocked in a 0.3% BSA/0.1% Triton X-100 solution, then incubated in primary antibody overnight at 4°C, washed in PBS and left in secondary antibody (Life Technologies) for a further 1 h at room temperature. Stained cultures were analyzed on a confocal fluorescence microscope (Zeiss LSM510).2, 1 mM MgCl2 10 mM glucose and 10 mM HEPES) was constantly perfused at 1–1.5 ml/min at room temperature. Whole-cell recordings were obtained from dsRed-positive neurons (SCRAM or SK1 KD) or neurons (rSK1-HA and rSK2-myc) using a K-methyl-sulfate based internal solution containing (mM): 135 KMeSO4, 5 NaCl, 10 Hepes, 2 Mg2-ATP, 0.3 Na3-GTP, 0.1 spermine and 7 phosphocreatine .Rat hippocampal neuronal cultures were recorded at least 2 weeks after viral infection (DIV 15–17). Coverslips were transferred from the incubator to the recording chamber, located on a stage of a fluorescence microscope (Zeiss 710). Ringer solution  to +10 mV for 100 ms was used to increase Ca2+ influx through voltage-gated Ca2+channels, which are necessary to activate IAHP , D-(−)-2-Amino-5-phosphonopentanoic acid  and 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt  were added to the ringer solution to reduce spontaneous activity and isolate mAHP and IData were collected using Axograph X software and a Multiclamp 700B amplifier (Molecular Devices). Signals were filtered at 10 kHz and digitized at 50 kHz using an ITC-16 A/D converter (InstruTech).n = 3, p = 0.002), but other higher molecular weight species were also greatly reduced . As shown previously . Next, expression of SK2 protein was tested using immunocytochemistry . We first tested the effect of overexpression of rSK1 or rSK2 on mRNA levels, using qPCR. Increasing the volume of rSK1 or rSK2 expressing virus added to neuronal cultures increased the relative expression of mRNA of the specific gene delivered without affecting the other: infection of 1 × 10hemistry . Transduhemistry  or rSK1-hemistry  showed chemistry , consisthemistry , its dishemistry .n = 3, p = 0.04; n = 5, p = 0.001; n = 4, p = 0.006; To test for the presence of rSK2 in the plasma membrane we again turned to the surface biotinylation assay to separate the membrane compartment. rSK1-HA and rSK2-Myc were transduced using lentivirus. As in Cosm6 cells , we founAHP), and in some types of neurons they are also present at excitatory synapses where they modulate synaptic strength , but the total membrane rSK2 tetramers (>200 kDa) appeared to be higher than the scrambled control  and rSK2 tagged with Myc (rSK2-Myc), in agreement with previous studies (D’Hoedt et al., AHP in infected rat neurons. Thus, by increasing or reducing the amount of rSK1 channels expressed in a heterologous system or rat neurons, we can decrease or increase rSK2 channels expressed on the cell membrane.These results in Cosm6 cells were confirmed in rat hippocampal neurons. Co-expression of rSK1-HA and rSK2-Myc resulted in an overall reduction in the amount of rSK2-Myc trafficked to the membrane. Importantly, expression of exogenous rSK1-HA in cultured neurons also resulted in a reduction in the total endogenous rSK2, showing that expression of rSK1 can modify the levels of membrane rSK2 channel. Finally, we show that by reducing the amount of rSK1 channels using RNAi, increases the amount of endogenous rSK2 channels expressed in the cell membrane, and is consistent with the finding that transgenic mice lacking SK1 show no overall effects on hippocampal neuronal electrophysiology (Bond et al., Previous studies have shown that when expressed in HEK293 cells, the interaction of rSK1 and rSK2 channels resulted in an overall larger SK-channels mediated current and a change in their pharmacology (Benton et al., Our results suggest that in the rodent brain rSK1 channels, rather than acting as independent membrane ion channels, are involved in trafficking of rSK2 channels to the plasma membrane. hSK1, which is ~84% homologous to the rodent channels (D’Hoedt et al., This was approved by the University of QLD Ethics Committee.EA discussed experiments, did experiments, and wrote the manuscript. PSe did experiments and wrote the manuscript. LX made virus. MR did experiments and made figures. AT did experiments, made figures and wrote the manuscript. PSa designed experiments, made figures and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Given these aspects, the search for new compounds based on floristic biodiversity is crucial. In the present work, we report the isolation, characterization and antileishmanial activity of six related neolignans  were performed using promastigote and amastigote forms of Leishmania (L.) infantum.Dried and powdered twigs of 1–6. Neolignans 2, 4 and 6 showed potential against amastigote forms of L. (L.) infantum , while compounds 1 and 3 were inactive. As neolignans 2–4 are chemically related, it may be suggested that the presence of the methoxyl group at C4 constitutes an important structural aspect to increase antileishmanial potential against amastigote forms. Compound 6, which consists of a methylated derivative of compound 5 (inactive) showed antileishmanial activity similar to that of the standard drug miltefosine (EC50 = 16.9 μM) but with reduced toxicity . Finally, two related monomers, eugenol and methyleugenol, were also tested and did not display activity, suggesting that the formation of dimeric compounds by oxidative coupling is crucial for antiparasitic activity of dimeric compounds 2, 4 and 6.Dereplication procedures followed by chemical characterization of isolated compounds by NMR enabled the identification of related neolignans 6 against L. (L.) infantum amastigotes as a scaffold for future design of new compounds for drug treatment of visceral leishmaniasis.This study highlights compound  Leishmania and transmitted by the bite of infected sandflies. The World Health Organization estimated 1.3 million new cases annually, divided into 300,000 cases of visceral leishmaniasis and 1 million cases of cutaneous leishmaniasis. -4,5-dimethoxybenzene (2), 1-[(7R)-hydroxy-8-propenyl]-3-[3′-methoxy-1′-(8′-propenyl)-phenoxy]-4-hydroxy-5-methoxybenzene (3), 1-[(7R)-hydroxy-8-propenyl]-3-[3′-methoxy-1′-(8′-propenyl)-phenoxy]-4,5-dimethoxybenzene (4), dihydrodieugenol (5), and dehydrodieugenol dimethyl ether (6) as shown in Fig. The antileishmanial potential of terature , allowinL. (L.) infantum to determine their EC50 values, followed by comparison of the values obtained for the standard drug (miltefosine). Furthermore, the cytotoxicity values (CC50) against NCTC – L929 cells as well as their in vitro selectivity index (SI) were determined. Promastigotes of L. (L.) infantum were incubated with compounds 1–6 for 48 h and the viability was determined by the MTT assay [4 and 6 were active, displaying EC50 values of 65.4 μM  and 88.8 μM , respectively. When tested against intracellular amastigotes, compounds 2, 4 and 6 showed activity with EC50 values of 57.9 μM , 67.7 μM  and 13.7 μM , respectively. Compounds 1, 3, 5 and monomers (eugenol and methyl eugenol) showed no activity against either tested form at the maximum tested concentration (100 μM). Miltefosine was used as standard drug with EC50 values of 16.9 μM (95% C.I. = 11.6 to 18.6 μM) against promastigotes and 16.4 μM (15.5 to 17.5 μM) against intracellular amastigotes . The standard drug miltefosine showed a CC50 value of 122 μM. Therefore, calculated SI values against amastigote forms were >  3.4, > 2.9 and >  14.6 for compounds 2, 4 and 6 respectively.All isolated compounds were evaluated against promastigotes and amastigote forms of TT assay , 19, 20.6 was similar to miltefosine. Recently, the DNDi (Drugs for Neglected Diseases initiative) defined criteria and proposed cutoff values to qualify the compounds investigated into hit compounds. They include: EC50 < 10 μM, SI tenfold more active vs. mammalian cell line, no structural alerts , chemical tractability  etc. [6 conforms with many of these criteria and could be eventually be addressed to the hit-to-lead optimization phase. Although the activity of compound 6 against promastigote forms of L. (L.) amazonensis has previously been reported in the literature [6 against intracellular amastigotes of L. (L.) infantum, the clinically relevant form of Leishmania, which results in a fatal visceral disease if untreated [It is important to highlight that the efficacy and toxicity of compound ps) etc. . Compounterature , the prentreated .1–6 and eugenol/methyleugenol, it was possible to suggest that coupling of monomers is a crucial step to obtain antileishmanial compounds since only dimeric derivatives (neolignans) displayed activity. By analyzing the activity data of closely related compounds 1–4, it was revealed that compounds 2 and 4 were active, suggesting that the presence of a methoxyl group at C-4 constitutes an important chemical feature to enhance the antiparasitic potential. As observed, neolignans containing free hydroxyl group at C-4 (1 and 3) were inactive, suggesting that polarity directly affects the antileishmanial potential of related compounds. Similarly, the presence of an additional hydroxyl group in the allyl side chain, as observed in compounds 2 and 4, causes a reduction in the potential. Comparing the activity of neolignans 5 and 6, it was observed that the presence of additional methoxyl group also enhances the antileishmanial activity as displayed by compounds 1–4, suggesting that the presence of this substituent is crucial to antileishmanial activity of these related neolignans. Finally, as previously reported [1–6 indicated that compound 6 was predicted as a non pan-assay interference compound (PAINS) and displayed some favorable in silico ADMET properties - non-mutagenic, non-carcinogenic, non-genotoxic, weak hERG blockers, with an acceptable volume of distribution (1.66–3.32 L/kg), and low rodent oral toxicity (LD50 = 810–2200 mg/kg).Comparing the data shown in Table reported , in sili


O respiration and residual oxygen consumption.Mitochondria are involved in generating more than 90 percent of cellular energy and are responsible for many cellular processes such as metabolism, cell signalling, apoptosis and ageing. Currently, there are a number of different experimental approaches employed to measure mitochondrial health and function. Here, we demonstrate a novel approach that quantifies substrate induced mitochondrial respiration from Drosophila. This protocol is optimized for mitochondria isolated from third instar larvae, and can also be used for mitochondria isolated from adult thoraces. This procedure outlines how to perform high throughput and high resolution mitochondria specific measurements for state II, state III, state IV The primary goal of this study is to develop a simple protocol that can be used to determine the basic bioenergetics functions of mitochondria isolated from Drosophila flies using the XF analyser (Seahorse Bioscience). Previous publications have developed protocols for cell lines ,2 and miTo date, two different experimental approaches have been employed to measure mitochondrial health and function in flies. The traditional Clark-type oxygen electrode is based on oxygen dissolved in liquid or gas phase in the sample chamber and signal is detected by polarography . Clark-tHere, we utilize the XF24 analyzer to measure mitochondrial function in Drosophila through determination of the oxygen consumption rate (OCR). The Seahorse XF extracellular flux analyzer is based on detection of OCR with oxygen-sensing fluorophores and extracellular acidification rate (ECAR) with pH sensor simultaneously in the same population of intact cells or isolated mitochondria . SeahorsDrosophila melanogaster larvae, which can also be used in adults. In this protocol, we assessed mitochondrial respiration by adding four injection compounds. The basal respiration (State II) is monitored before addition of ADP. Basal respiration is controlled by proton leak and substrate oxidation. The respiration of state III was assessed by injection of ADP to stimulate ATP turnover. State III respiration reflects OXPHOS activity under saturated substrate, oxygen and ADP levels. State III respiration can be terminated by adding the ATP synthase inhibitor oligomycin, to obtain a state IVo rate. In the presence of oligomycin, respiration is highly dependent on proton leak [Here we outline a protocol optimized for mitochondrial extracts from ton leak . The injton leak ,14. Fina•2 of cottonOne-cc syringe filled with 5.0 cm•Plastic microtube pestle (Dounce)•Bradford reagent (Sigma B6916)•1 mg/ml protein standard (Sigma P0914)•XF24 FluxPak NOTES: The XF24 FluxPak contains 18 XF24 extracellular flux assay kits. Each assay kit contains one sensor cartridge, one utility plate , and one lid. The XF24 FluxPak includes 20 polystyrene XF cell culture microplates for mitochondria seeding purpose and one bottle of 500 ml of XF24 calibrant solution (pH7.4).•Isolation buffer: 154 mM KCl, 1 mM EDTA, pH 7.4•2PO4 (Sigma P9791), 2 mM MgCl2 (Sigma M1028), 3 mM HEPES (Sigma H0887), 1mM EGTA (Sigma E4378), FA-free BSA 0.2% (A7511), adjust solution to pH 7.2 using 5 N KOH.Mitochondrial Assay Solution (MAS): 115 mM KCl (Sigma P9333), 10 mM KHCAUTIONS: The presence of BSA in the MAS solution preserves mitochondrial coupling. It is essential to have a concentration of 0.2% BSA in the MAS solution.•Injection compounds: Port A: 2.5–20 mM ADP (Sigma A2754); Port B: 40 µM Oligomycin (Sigma O4876); Port C: 2.5−320 µM BAM 15 (Tim Tec ST056388)/ 40−160 µM FCCP (Sigma C2920); Port D: 20 µM Antimycin A (Sigma A8674) and Rotenone (Sigma R8875).TIPS: ADP can be prepared as 100 mM stock in MAS solution and adjusted to pH 7.2. Other injection compounds can be prepared at 1000 × in DMSO and diluted to 10 × with MAS for port loading. The final concentration in the well after injection is 1 × concentration.•Complex I Assay Media: 11 mM Pyruvate (Sigma P2256), 11 mM malate (Sigma M1000), 11 mM L-proline (Sigma P0380), pH 7.2.TIPS: Pyruvate, malate and L- proline can be prepared as 100 mM stock in MAS solution and adjusted to pH 7.2. The substrates can then be diluted to the desired concentration (11 mM) with MAS.•pH meter•Eppendorf centrifuge 5810R pre-cooled to 4°C•Plate reader (SpectraMax M3)•2 incubator set to 15°CNon CO•XF24 Extracellular flux Analyzer  with the run temperature set as appropriate (we used 23°C).1.1.1.Collect 10 whole wandering third instar larvae and wash them in water and 70% ethanol . Place them in 100 µl of ice-cold mitochondrial isolation buffer in 1.5 ml microcentrifuge tubes.1.2.Gently homogenize the third instar larvae or thoraces with 80 strokes up and down with a plastic microtube pestle.Mitochondrial isolation for DrosophilaCAUTION: Press dounce straight up and down without grinding. If a pellet forms during homogenization, release it with a spatula or pipette tip and continue the process.1.3.Transfer the homogenate into a 1-cc syringe containing 200 µl of isolation buffer.1.4.Force the homogenate through the cotton filter and collect in a new 1.5 ml microcentrifuge tube.1.5.Centrifuge the filtered homogenate at 1500 × g at 4°C for 8 min.CAUTION: Centrifugation at a higher speed should be avoided as increasing the spin to 5000 × g results in mitochondria with low respiration rates.1.6.Remove and discard the cloudy supernatant, be careful not to disturb the pellet.1.7.Wash the pellet with 200 µl of mitochondrial isolation buffer and then resuspend in 20 µl of isolation buffer.1.8.Perform the Bradford assay according to the manufacturer’s instructions (Sigma B9616).TIPS: We suggest 2 µl of mitochondrial stock be diluted with 10 µl of mitochondrial isolation buffer (1 to 6 dilutions) to ensure the readings are within the standard curve range for Bradford assay.2.2.1.The day before the assay, hydrate the XF sensor cartridge at 23°C overnight in XF calibration buffer.Prepare the XF sensor cartridgeNOTES: The assay was performed at 23°C and temperature is subjective to change based on the study design.2.2.On the day of assay, load the XF sensor cartridge injections port with injections compounds.2.3.Volume of compounds loaded in each port: Port A: 50 µl; Port B: 55 µl; Port C: 60 µl; Port D: 65 µl.2.4.Table 1).Calibrate the sensor cartridge by following the XF instrument protocol  with MAS to yield a final concentration of 0.02−0.4 µg/µl.Preparation of mitochondrial complex I assay plateTIPS: Due to the high concentration of mitochondria in the suspension, it is recommended that the mitochondria be first mixed by gently pipetting, and then performing 1 to 6 dilutions with MAS to form a final volume of ~100µl . This is then added to a larger volume to form the desired concentration.3.2.E.g., 50 µl of diluted mitochondria with 0.1, 0.2 and 0.4 µg/uL concentration will results in 5, 10 and 20 µg of total mitochondria per well.Pipette 50 µl of the diluted mitochondria into each well of XF cell culture microplate. 3.3.Transfer 50 µl of MAS into each background correction wells.3.4.Centrifuge the XF cell culture microplate at 2254 × g at 4°C for 20 min.3.5.After centrifugation, visualize the mitochondria under the microscope using 20 × magnification. Make note of any wells that do not have a “monolayer” of mitochondria adhered to the well bottom.3.6.Add 450 µl of complex I assay media to each well and incubate the cell culture microplate at 23°C for 10 min.NOTES: The assay was performed at 23°C and temperature is subjective to change based on the study design.3.7.Table 1).Subsequently, exchange the calibration plate with the cell culture microplate and follow the instructions on the instrument controller to continue with XF instrument protocol . However, the optimum RCR values can be obtained using 5 and 10 µg of mitochondria. We recommend using 10 µg of mitochondria as the state II OCR falls within the range 50 pmoles.min-1< OCR < 200 pmoles.min-1. For the subsequent titration of the injection compounds, 10 µg of mitochondria was used in all wells.The typical microscopy image for the mitochondrial “monolayer” is shown in Figure 2A, the concentration of ADP is saturated at 1 mM concentration. ADP injection after 1 mM will not stimulate higher state III OCR. The optimum final well concentration for BAM 15 was 16 µM and for FCCP was 8 µM . Increasing the concentration of BAM 15 above the saturation point had no effects on maximum respiration. In contrast, an increase in FCCP above 8 µM reduces the maximum respiration. The results indicate that FCCP is cytotoxic to the mitochondria (above 8 µM) and higher concentrations depolarize the mitochondrial membrane [As shown in This protocol was originally optimized for mitochondria extracted from third instar larvae. The optimized protocol for complex I mitochondrial respiration consists of 10 µg mitochondrial protein with the following injection for each port, Port A: 10 mM ADP; Port B: 40 µM oligomycin; Port C: 160 µM BAM 15 or 80 µM FCCP; Port D: 20 µM rotenone and antimycin A. Subsequently, we have been successful in applying this protocol to mitochondria extracted from adult thoraces after reducing the mitochondrial concentration to 5 µg.Following initial preliminary studies, this final protocol was optimized in four steps  in four separate plates. Where possible, we recommend researchers run each experimental treatment with at least four biological replicates each with three pseudo-replicates. Finally, to determine robustness of the method we replaced complex I assay media with 11 mM of succinate (Sigma S2378) and 11 mM of glycerol-3-phosphate (Sigma G7886). The basic protocol successfully determined complex II functions from mitochondria extracted from third instar larvae and complex III functions from mitochondria extracted from adult thoraces, respectively. Intriguingly, complex III functions from mitochondria extracted from third instar larvae and complex II functions from mitochondria extracted from adult thoraces failed suggesting a biological basis.Table 2.Tips for troubleshooting can be found in


Breast cancer (BC) cells (BCCs) can retain cellular quiescence for decades, a phenomenon referred to as dormancy. BCCs show preference for the bone marrow (BM) where they can remain dormant for decades. Targeting BCCs within the BM is a challenge since the dormant BCCs reside within BM stroma, also residence for hematopoietic stem cells (HSCs). Dormant BCCs could behave as cancer stem cells (CSCs). The CSCs and HSCs are similar by function and also, by commonly expressed genes. The method by which dormant BCCs transition into clinically metastatic cells remains unclear. This study tested the hypothesis that macrophages (MΦs) within BM stroma, facilitates dormancy or reverse this state into metastatic cells. MΦs exhibiting an M2 phenotype constitute ~10% of cultured BM stroma. The M2 MΦs form gap junctional intercellular communication (GJIC) with CSCs, resulting in cycling quiescence, reduced proliferation and carboplatin resistance. In contrast, MΦs expressing the M1 phenotype reversed BC dormancy. Activation of M2a MΦs via the toll-like receptor 4 (TLR4) switched to M1 phenotype. The switch can occur by direct activation of M2a MΦs, or indirectly through activation of mesenchymal stem cells. M1 MΦ-derived exosomes activated NFкB to reverse quiescent BCCs to cycling cells. Using an in vivo model of BC dormancy, injected Mi MOs sensitized BCCs to carboplatin and increased host survival. In summary, we have shown how BM stromal MΦs, through exosomes, regulate the behavior of BCCs, by either inducing or reversing dormancy. Disseminated BCCs can enter the bone marrow (BM) long before detection4. This allows for the establishment of BC dormancy before clinical diagnosis, in addition to transition into cellular quiescence during the clinical course of the disease7. As compared to micrometastasis in sentinel lymph nodes, BC metastasis to the BM leads to a worse prognosis8. BM stromal cells form a critical niche for BCCs to survive. The stromal cells facilitate BCC quiescence, immune escape, changes in cytokine production and gap junctional intercellular communication (GJIC)10.Breast cancer (BC) cells (BCCs) may exist in cellular quiescence (dormancy) for decades10. Nonetheless, an understanding of how BM stroma support BCC dormancy is important since the same stromal cells can also cause BC resurgence13. BM stroma is comprised of several cell types such as macrophages (MΦs), fibroblasts, osteoblasts, mesenchymal stem cells (MSCs), and adipocytes14.Precise targeting of dormant BCCs in BM is a challenge. The quiescent BCCs have stem cell-like properties, and share similarities with endogenous hematopoietic stem cells (HSCs). The anatomical location of the cancer cells with HSCs makes it difficult to target the dormant BCCs without untoward effects on the hematopoietic system17. M2 MΦs are classified as M2a, M2b, M2c, or M2d and such designation, depends on the mode of activation16. M1 MΦs elicit a proinflammatory response and M2 MΦs, immune suppression, wound healing, and angiogenesis17. The biological function of a particular MΦ type may be influenced by the surrounding niche, such as MSCs within BM18. We tested the hypothesis that activation of stromal cells causes one of its component, M2 MΦ, to polarize into the M1 phenotype to reverse dormant BCCs into proliferating cells.MΦs are broadly divided into nonactivated, classically activated (M1) and alternatively activated (M2) types21. TLR4 is a member of the pattern recognition receptor (PRR) system, which can be stimulated by microbiome-derived ligands such as lipopolysaccharide (LPS). TLR4 can also bind to other pathogen associated molecular pattern and endogenous damage-associated molecular patterns (DAMPs)22.This study activated toll-like receptor 4 (TLR4) on MΦs to study how this influence BC behavior because TLR4 has been linked to cancer recurrence10. In the presence of M1 MΦs, the majority of otherwise chemoresistant CSCs were responsive to carboplatin. Injection of M1 MΦs into immune deficient mice harboring dormant BCCs reversed dormancy resulting in the BCCs becoming sensitive to carboplatin. The mice injected with M1 MΦs showed prolonged survival with no evidence of the dormant BCC. In contrast, mice injected with M2a MΦs survived, but with persistence of the dormant BCCs. The data are discussed in the context of how BCCs may react to PRR stimulation, and the potential risk for long-term BC survivors.We report on conversion of M2 MΦs into M1 MΦ phenotype by LPS. Such conversion occurred directly on M2 MΦs and indirectly, through MSCs. The M1 MΦs secrete exosomes, which reversed the quiescent phase of BCCs, particularly the cancer stem cell (CSC) phenotype without affecting their “stemness”The use of human blood and BM aspirates was approved by the Institutional Review Board (IRB) of Rutgers, Newark Campus. All subjects ranged were 18–35 years and signed the informed consent forms. The Institutional Animal Care and Use Committee (IACUC) approved the use of mice. The studies presented in this study adhered to the approved protocols.23. Total BM aspirate with nucleated cells (107) were added to 25-cm2 tissue culture flasks  in stromal media. At day 3, the red blood cells and neutrophils were removed by Ficoll–Hypaque density gradient and the mononuclear cells retrieved from each flask were re-added in fresh stromal medium. The flasks were incubated until confluence with a weekly replacement of 50% fresh stromal medium.Stroma was prepared from BM aspirates as described24. The recovered MΦs were incubated in Teflon jars for 2 h to allow any degraded membrane protein to be re-expressed. In five analyses with stroma from a different donor we observed 10 ± 1.3% (±SD) CD206+/MHC-II-cells. Indeed, MΦs, which are part of the stromal compartment, are important for functional hematopoiesis25.Confluent stromal cells were assessed for MΦ phenotype by immunofluorescence using anti-CD206 (M2) and anti-MHC-II (M1). MΦs are trypsin resistant and de-adherence requires treatment with 0.2% collagenase28. Monocytes were selected by incubating the mononuclear cells with Dynabeads Flow Comp human CD14+ kit from Invitrogen . This resulted in cells that were positive for nonspecific esterase and >99% CD14+ cells by flow cytometry  were incubated for 48 h in 6-well plates with RPMI 1640 containing 10% FCS and 50 ng/mL of M-CSF29. The media were replaced with fresh media containing 10 ng/mL IFNγ for M1 MΦ and 20 ng/mL IL-4 for M2a MΦ. MΦs within BM stroma were ascribed the designation of M2 and were CD206+ and MHCII−.The culture of M1 and M2a MΦs was performed as described using human peripheral blood mononuclear cells (PBMCs) and BM mononuclear (BMNCs)try Fig. . The monBCCs were synchronized for 72 h in serum-free media supplemented with 1× insulin–transferrin–selenium. At synchronization, the cells were challenged with exosomes by replacing the media containing 10% FCS.The assessment of live/dead cells, referred as cell vitality, used two different dyes from Molecular Probes. The live cells can convert a redox dye (resazurin) into a fluorescent end product (C12-resorufin). SYTOX green dye is a permeable cell stain that is used to measure either injured or dead cells. Cells were incubated with 500 nM C12-resazurin and 10 nM SYTOX in 100 µl 1× phosphate-buffered saline (PBS) for 15 min at 37 °C. Cell suspension was diluted with 400 µl 1× PBS. Stained cells were immediately analyzed on FACSCalibur (excited at 488 nm and measured fluorescence emission at 530 and 575 nm). Cell populations were gated into three groups—live, injured, and dead, and then quantified.g as described12. The remaining particles were pelleted by ultracentrifugation  at 100,000g for 18 h. Vesicles expressing CD63 were immunoselected using CD63 magnetic bead isolation. The recovered particle size was verified by nanoparticle tracking analysis (NTA) using a NanoSight NS300 instrument  as described11. The data were analyzed with the NTA software (NANOSight version 2.3) using dilutions with deionized water. Three videos at a minimum of 200 completed tracks were collected at 30-s time intervals/video per sample.Exosomes were isolated using a multi-step process. Large vesicles were eliminated by sequential centrifugation up to 50,000Exosomes were further distinguished by flow cytometry with magnetic beads coupled to anti-CD9-PE, anti-CD63-FITC and anti-ALIX-APC (BD Biosciences).hi MDA-MB-231 or T47D (103 in 0.2 mL 1× PBS), as described10. To ensure dormancy mice were injected intraperitoneally with low dose of carboplatin (2 mg/kg) at days 2 and 5, resulting in Oct4hi BCCs in the endosteal regions of femurs. At day 7, the mice were divided into groups of ten, and injected intravenously with (a) 106 CMTMR-labeled M1 MΦ in 0.2 ml 1× PBS; (b) 106 CMTMR-labeled M2a MΦ in 0.2 mL 1× PBS; (c) Control, injected with 0.2 mL 1× PBS. Each experiment was repeated three times. At days 10, 12, and 15, the mice were injected with 5 mg/mL carboplatin. At day 20, the mice were euthanized and the femurs and lungs were harvested. The femurs were washed to remove the cells and the endosteal region scraped and immediately analyzed for fluorescent cells. Femurs were also decalcified, embedded in paraffin and then sectioned at the digital imaging and histology core facility at New Jersey Medical School. Sections from the femurs of mice were subjected to immunohistochemistry for M1 and M2 MФs  were purchased from Taconic Farms  and housed in an AAALAC-accredited facility. BC dormancy in BM was established by tail vein injection of Oct4 MФs see  In separ11. The anti-miRs were purchased from Life Technologies—Applied Biosystems . The MSCs were washed and then resuspended in PBS for injection into nude mice.MSCs were co-transfected with anti-miR-223 and anti-miR-222 or negative control anti-miR using Lipofectamine RNAiMAX reagent  as described10. Another group was injected with PBS for the purpose of assessing MФ type in femurs. The dormant group was further subdivided into two groups: Group 1 was injected 106 MSCs transfected with anti-miR222/223 and Group 2, with control anti-miR. The anti-miRs can enter the dormant BCCs to reverse dormancy into proliferating BCCs11.BC dormancy was established in female BALB/c nude mice (6 weeks) old as described, also explained abovep value ≤ 0.05 was considered to be significant. The significant effects of exosomes and media were deduced by p values of <0.001 in two-way ANOVA with Bonferroni post-test that compared cycling progression, G0/G1 vs. S-phase.The data were analyzed using one-way ANOVA with Bonferroni’s multiple comparison test as the post hoc test or two-way ANOVA with Bonferroni correction. A 31. Western blots with whole cell extracts identified the marker of M2 MФs, mannose receptor C-type1 (MRC1/CD206), within stroma, similar to extracts from M2a MΦ control  reduced in stroma as compared to control M2a MΦs 33.In order to include all components of BM stroma to understand BC dormancy, nonhematopoietic cells and MΦs, stromal cultures are established with BM aspiratesrol Fig. 32. The nMΦs Fig. , consist35  decrease of IL-10 whereas M1 to M2 MΦ increased IL-6 and IL-12  higher for M2 MΦs and similar for Cx26  Fig. . The dyep < 0.05) increased BCC proliferation as compared to M2a MΦs  decrease in G1 phase and increase in S/G2 phases by M1 MΦs , co-cultured for 72 h, showed significantly  in 72-h co-culture with M1 or M2 MΦs indicated no effect on self-renewal after fiver serial passages. Representative spheres are shown from passage 5  were mostly in G0 phase with M2a MΦs  reduced the percentage of G0 cells  or without exosomes. A representative image for MDA-MB-231 is shown for time 0 up to 50 h  of the scratch assay indicated a significant (p < 0.05) increase in migration with exosomes from M1 as compared to M2 /MET. Migration using scratch assay with CSCs, mostly in G0 h Fig. . The plo M2 Fig. .Fig. 6MiWestern blots for vimentin and E-cadherin were increased in MDA-MB-231 exposed to exosomes from M1 and M2 MΦs. This suggested mixed behavior of BCCs with respect to EMT and MET Fig. . There w41. We asked if M1-derived exosomes can induce BCC cycling via active NFκB. Functional studies were needed because phosphorylated (phospho) p65 in the cytosol may not be active42. Serum-starved BCCs and nontumorigenic HBL-100 cells were treated with exosomes from M1 or M2 MΦs. Flow cytometry showed increase of p65 in all cell lines but only M1 MΦ-derived exosomes induced TLR4 on the BCCs (~98% vs. <4% on HBL-100)  Fig. , suggest00) Fig. . To furt00) Fig. .Fig. 7TL43. Exosomes from M1 or M2 MΦs were added and 24 h later, the cells were analyzed for cycling phase by PI labeling. PDTC only blunted cycling of M1-derived exosomes for MDA-MB-231  for 24 h231 Fig. , suggest0/G1 phase, were incubated with 4 × 107 exosomes from M2a or M1 MΦ, followed by treatment with 50 μg/mL of carboplatin. After 72 h, flow cytometry for the CSC health indicated significant  cell death by M1 MФs  in the cellular region of lungs  in mice treated with M1 MΦs but undetectable CSCs with M2 injection Fig. . The latAt 20 days after the last treatment, we examined scraped sections of femurs with M1 MΦs, treated or untreated with carboplatin. Only red fluorescent cells were noted with carboplatin, indicating elimination of CSCs with Oct4A-GFP Fig. , left. In = 10/group) for mice injected with M1 MΦs without carboplatin succumbed by day 22  by activated and inactivated MSCs. We are currently identifying the candidate secretome(s) involved in determining how the BM microenvironment sustain a particular MΦ type.In vivo, M1 MΦs sensitized CSCs to carboplatin Fig. . The exp51.It is interest that M1 MΦ-derived exosomes induced TLR4 in BCCs Fig. . The TLRUpon synchronization, BCCs showed similar effects on cycling quiescence as the studies conducted with CSCs in the presence of MΦs. This suggested that MΦs may affect any BCC within an environment that promotes survival and reduced cycling. Thus, it is likely that dormant BCCs might be heterogeneous with respect to relative maturity. These findings have added to the general information on BM stromal support of BC survival. The data in this report have provided information that would lead to omics studies for the purpose of understanding how dormancy and cancer recurrence occur. We are in the process of comparing the exosomes from M1 and M2 MΦs. These analyses are insufficient since the preliminary evidence suggest that proteins within the exosomes might also be responsible (Unpublished). The contents of the exosomes involved in these studies are included in an independent paper, in preparation for publication.11 (Fig. We have determined the MΦs in healthy and tumor-bearing mice using a model of dormancy and reverse dormancy11 Fig. . M2 and Supplemental methods and figures


The obtained material had a SO3H density of 0.64 mmol·g−1 and a specific surface area of 602 m2·g−1. The catalytic properties of AC-SO3H were compared with that of two commercial solid acid catalysts, Nafion NR50 and Amberlyst-15. In a 10-h esterification reaction of acetic acid with ethanol, the acid conversion with AC-SO3H (78%) was lower than that of Amberlyst-15 (86%), which could be attributed to the fact that the SO3H density of the sulfonated carbon was lower than that of Amberlyst-15 (4.60 mmol·g−1). However, AC-SO3H exhibited comparable and even much higher catalytic activities than the commercial catalysts in the esterification of aliphatic acids with longer carbon chains such as hexanoic acid and decanoic acid, which may be due to the large specific surface area and mesoporous structures of the activated carbon. The disadvantage of AC-SO3H is the leaching of SO3H group during the reactions.Sulfonated (SO Hara’s group first prepared sulfonated carbon catalysts via the sulfonation and carbonization of polycyclic aromatic hydrocarbons [3H group density. Carbon-based solid acid catalysts could also be obtained by sulfonation of carbon nanotubes [Sulfonated , a fact attributed to the chemical inertness of AC [et al. reported the generation of a sulfonated carbon material with a SO3H density of 0.44 mmol·g−1 by treatment of AC with concentrated H2SO4 [2-impregnated wood powders [Activated carbon (AC) is widely used as a catalyst support in a variety of industrial and environmental applications for its chemical stability, high specific surface area, and low cost . Studiesed H2SO4 ,14. The 2SO4 treatment method, chemical reduction of aryl diazonium salts has also been proved to be quite efficient for the functionalization of carbon materials [et al. reported the preparation of carbon-based solid acid catalysts by covalent attachment of SO3H radicals on the surface of ordered mesoporous carbon in a chemical reduction process [3H-containing group by reacting AC with 4-benzenediazonium sulfonate using hypophosphorous acid (H3PO2) as reducing agent. The structure and catalytic properties of the obtained material were also examined.Besides the Haterials . Feng et process ,18. Howe3H). The broad C (002) diffraction peak (2θ = 15-30º) can be attributed to the amorphous carbon structures. The weak and broad C (101) diffraction peak (2θ = 40-50º) is due to the a axis of the graphite structure [3H, suggesting that the chemical reduction process does not affect the microstructure of carbon materials.eak 2θ = 5-30º caneak 2θ = 0-50º is tructure . There i3H density of sulfonated carbon material was determined by combining the S elemental analysis and the total acid site density results. As shown in −1, respectively. The total acid density of AC increased from 0.30 to 1.01 mmol·g−1 via the sulfonation.The SO−1) was close to that of the total acid density (0.71 mmol·g−1) in the sulfonation process. Therefore the SO3H density of sulfonated AC is about 0.64 mmol·g−1. X-ray photoelectron spectroscopy (XPS) analysis was conducted to further verify the chemical state of sulfur in AC-SO3H. As shown in 3H group [These results showed that the increment of S content (0.64 mmol·g3H was studied by TGA results (under flowing N2). As shown in 3H below 150 ºC was similar to that of AC. However, the thermogravimetric curve of AC-SO3H at higher temperatures showed a more significant weight loss compared to that of AC, which is mainly due to the gradual desorption and thermal decomposition process of PhSO3H group.The thermal stability of AC-SO2 adsorption isotherms of the carbon materials. Both AC and AC-SO3H exhibit similar isotherms with evident hysteresis loops in the relative pressure range of about 0.4-0.9. The surface properties of the carbon materials are summarized in 2·g−1 and 0.47 cm3·g−1 to 602 m2·g−1 and 0.38 cm3·g−1, suggesting that a small part of the pore space has been occupied by the grafted SO3H groups. The pore diameter does not change during sulfonation (from 2.5 to 2.4 nm).3H has been investigated in the esterification of three kinds of aliphatic acids with different carbon chain lengths , as shown in 3H gives an acid conversion of 78%. The conversion with Nafion is 61%. As shown in 3H density of Amberlyst-15 (4.60 mmol·g−1) is much higher than that of AC-SO3H (0.64 mmol·g−1) and Nafion (0.45 mmol·g−1). Therefore, it may be conferred that the catalytic activity for acetic acid esterification is mainly dependent on the acid density of the catalyst. The catalytic activity of AC-SO3H is comparable in this case with that of Amberlyst-15. The advantage of our carbon catalyst becomes more evident in the esterification of decanoic acid, as shown in 3H (52%) is much higher than that with Amberlyst-15 (36%) and with Nafion (17%) after 10 h of reaction. The results in 3H has a large surface area (602 m2·g−1) and high density of mesopores, which may be more convenient for larger aliphatic acid molecules to adsorb on the catalyst surface and approach to the active acid sites. Therefore, our sulfonated carbon materials may have potential applications in the acid-catalyzed reactions of large organic molecules. 3H group on the carbon catalyst, AC-SO3H was reused in a series of acetic acid esterification reaction cycles. Before each cycle, AC-SO3H was washed with 1 mol·L−1 H2SO4, and then washed repeatedly with deionized water at least seven times until no SO42− ions were detected in the washed water [To investigate the stability of SOed water .3H density in the reaction cycles. The fresh catalyst has a SO3H density of 0.64 mmol·g−1. The value decreases evidently in the beginning four reaction cycles and then remains stable at 0.42 mmol·g−1 in the following three cycles. The results indicate that about 34% SO3H group (0.22 mmol·g−1) of the fresh catalyst will leach out during the reactions, while 64% SO3H is preserved. It has been pointed out elsewhere that the leaching of SO3H groups is a common problem for sulfonated catalysts [3H groups.atalysts . More efActivated carbon was purchased from the Taiyuan Xinhua Activated Carbon Company . Commercial solid acid catalysts, Amberlyst-15 and protonated Nafion NR50, were purchased from Alfa Aesar . All other reagents were in reagent grade and used without any further treatment. 3PO2) as the reducing agent [2  was added dropwise into the flask. The mixture was stirred for 50 min. The obtained white precipitate was filtered and washed with cold water. The sulfonated carbon material was obtained by the reaction of 4-benzene-diazonium sulfonate with activated carbon using hypophosphorous acid  was added and the temperature was controlled at 0-5 ºC. After stirring for 30 min, another portion of 50 wt % H3PO2 (100 mL) was added and the mixture was stirred for another 30 min. The sulfonated AC is filtered and washed with distilled water at least seven times and then with acetone, and dried at 80 ºC. To functionalize AC, distilled water (100 mL), ethanol (100 mL), diazonium salt (12.0 g) and AC (2.0 g) were added in a three-necked flask. Then 50 wt % H2 adsorption analysis (Micromeritics ASAP300), and TGA (Netzsch STA 449C).The sulfur content of carbon materials and commercial catalysts (Amberlyst-15 and Nafion NR50) was determined by a Sulfur Analyzer (KZDL-4) . The totThe catalytic properties of the sulfonated carbon material were examined through the esterification of a series of aliphatic acids  with ethanol at 70 ºC. Typically, aliphatic acid (0.1 mol), ethanol (1.0 mol), and the carbon catalyst  (0.2 g) were used in the reactions. The liquid reaction mixtures were analyzed by gas chromatography (Agilent GC 7890). 3H density of 0.64 mmol·g−1 and a specific surface area of 602 m2·g−1 was synthesized by sulfonating activated carbon in an aryl diazonium salt reduction process. In the esterification reaction of acetic acid with ethanol, AC-SO3H showed inferior catalytic activity to the commercial catalyst, Amberlyst-15, because the former’s SO3H density is much lower than the latter’s (4.60 mmol·g−1). However, AC-SO3H possessed evident advantages in the esterification of decanoic acid, which was probably due to the high specific surface area and a large number of mesopores of the activated carbon. Our study results suggest that AC-SO3H may have potential applications in the acid-catalyzed reactions of large organic molecules. The main disadvantage of AC-SO3H is the leaching of SO3H groups during the reactions. A porous carbon-based solid acid catalyst possessing a SO


Background: Thioredoxin (TRX) acts as both a scavenger of reactive oxygen species (ROS) and an immuno-modulator. Although quercetin has been shown to favorably modify allergic rhinitis (AR) symptoms, its influence on TRX production is not well defined. The present study was designed to examine whether quercetin could favorably modify AR symptoms via the TRX production of nasal epithelial cells in vitro and in vivo. Methods: Human nasal epithelial cells (HNEpCs) were stimulated with H2O2 in the presence of quercetin. TRX levels in 24-h culture supernatants were examined with ELISA. BALB/c male mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA every other day, beginning seven days after the final sensitization. The mice were orally administered quercetin once a day for five consecutive days, beginning seven days after the final sensitization. Nasal symptoms were assessed by counting the number of sneezes and nasal rubbing behaviors during a 10-min period immediately after the challenge. TRX levels in nasal lavage fluids obtained 6 h after the challenge were examined by ELISA. Results: Treatment with 1.0 nM quercetin increased H2O2-induced TRX levels. The oral administration of 20.0 mg/kg of quercetin significantly inhibited nasal symptoms after the challenge. The same dose of quercetin significantly increased TRX levels in nasal lavage fluids. Conclusions: Quercetin’s ability to increase TRX production may account, at least in part, for its clinical efficacy toward AR. Allergic rhinitis (AR) is a well-known type of chronic allergic inflammation that occurs in the nasal mucosa and is characterized by multiple symptoms such as sneezing, itching, and watery rhinorrhea ,2. AlthoAR treatment can be divided into three main categories: allergen avoidance, drug therapy, and immunomodulating therapy . AllergeQuercetin is a dietary flavonoid found in red wine, tea, many fruits, and onions . For man2 and H2O2 [It is currently accepted that inflammatory cells including eosinophils, which are the most important effector cells in the development of inflammatory diseases, produce several types of toxic granule proteins and reactive oxygen species (ROS), such as Oand H2O2 ,18. Althand H2O2 ,19. Convand H2O2 ,21. In aand H2O2 ,23. TRX and H2O2 ,23. HoweSpecific 5-week-old pathogen-free BALB/c male mice were purchased from CLEA Japan Co., Ltd. . The mice were maintained in our animal facility at 25 °C ± 2 °C with 55% ± 10% humidity under a 12-h dark/light cycle and were allowed free access to tap water and standard laboratory rodent chow  throughout the experiments. Each control and experimental group consisted of five mice. All animal experiments were approved by the Ethics Committee for Animal Experiments of Showa University (Approved No. 54011). Date of approval: 1 April 2018.3  were obtained from Sigma-Aldrich Co. Ltd. as preservative-free pure powders.Quercetin was purchased from Sigma-Aldrich Co., Ltd.  as a preservative-free pure powder. Quercetin was first dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10.0 mM. This solution was then diluted with Airway Epithelial Cell Growth Media  at appropriate concentrations for the experiments; then, the solution was sterilized by passing through 0.2-μm filters and was stored at 4 °C until use. To assess in vivo use, quercetin was mixed with 5% tragacanth gum solution at a concentration of 7.5 mg/mL . Chicken5 cells/mL and used as target cells. The HNEpCs (5 × 105 cells/mL) were stimulated with 12.5–100.0 μM H2O2 for 24 h in a final volume of 2.0 mL. To examine the influence of quercetin on TRX production, the HNEpCs (5 × 105 cells/mL) were stimulated with 50.0 μM H2O2 in the presence of 0.1–10.0 nM quercetin for 24 h in a final volume of 2.0 mL. To examine TRX mRNA expression, the cells were stimulated with 50.0 μM H2O2 in the presence of 0.1–10.0 nM quercetin for 12 h. Quercetin was added to the cell cultures 2 h before H2O2 stimulation.HNEpCs, purchased from PromoCell GmbH, were suspended in AECG medium (PromoCell GmbH) at a concentration of 5 × 105 cells/mL) were cultured with either 12.5–100.0 μM H2O2 or 0.1–10.0 nM quercetin for 24 h. The cells were then collected, and cell viability was assessed by the trypan blue dye exclusion test. The dead cells were stained with trypan blue, and the proportion of dead cells was determined by counting 300 total cells.HNEpCs (5 × 10+ mRNA was extracted from cultured cells with oligo(dT)-coated magnetic micro beads . The first-strand cDNA was synthesized from 1.0 μg of Poly A+ mRNA using a Superscript cDNA synthesis kit  according to the manufacturer’s recommendations. Polymerase chain reaction (PCR) was then performed using a GeneAmp 5700 Sequence Detection System . The PCR mixture consisted of 2.0 µL of sample cDNA solution (100 ng/µL), 25.0 µL of SYBR-Green Mastermix (Applied Biosystems), 0.3 µL of both sense and antisense primers, and distilled water for a final volume of 50.0 µL. The conditions used for the reaction was as follows: 4 min at 94 °C, followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. GAPDH was used as an internal control. TRX mRNA levels were calculated using the comparative parameter threshold cycle and normalized to GAPDH. The primers used for real-time RT-PCR were as follows: 5′-GCCTTGCAAAATGATTCAAGC-3′ (Sense) and 5′-TTGGCTCCAGAAAATTCACC-3′ (Antisense) for TRX [TRX mRNA expression was examined by the methods described previously 24). Briefly, Poly A. BrieflyBALB/c mice were sensitized with an intraperitoneal injection of 20.0 μg/mL OVA in phosphate-buffered saline (PBS) combined with 1.0 mg of alum in a total volume of 200.0 μL on days 0, 7, and 14 ,4. On daThe mice were anesthetized by intraperitoneal injection with 50.0 mg/kg sodium pentobarbital  6 h after the OVA nasal challenge. The trachea was exposed and cannulated to introduce 1.0 mL of PBS . The lavNasal allergy symptoms were assessed by counting the number of sneezes and nasal rubbing movements for 10 min immediately after the OVA nasal instillation. The experimental mice were placed into plastic animal cages (35 × 20 × 30 cm) for approximately 10 min to acclimate. After the nasal instillation of 0.1% OVA solution in PBS in a volume of 5.0 μL, the mice were placed into plastic cages , and the number of sneezes and nasal rubbing movements were counted for 10 min .The TRX levels in the culture supernatants and nasal lavage fluids were examined using human and mouse TRX ELISA test kits  according to the manufacturer’s recommendations. The minimum detectable levels of the ELISA test kits were 1.172 ng/mL and 0.078 ng/mL for humans and mice, respectively.The oxidative stress responses in the nasal mucosa were evaluated by measuring lipid peroxide levels in nasal lavage fluids using d-ROM tests  according to the manufacturer’s recommendations. The results were expressed as mean Carratelli Units (CARR U) ± SE.P value of less than 0.05 was considered significant.The statistical significance between the control and experimental groups was assessed with an ANOVA followed by Dunette’s multiple comparison test. A 2O2 stimulation could increase TRX production from HNEpCs and to determine the optimal concentration of H2O2 for stimulation. Thus, the cells were stimulated with various concentrations of H2O2 for 24 h, and the TRX levels in the culture supernatants were determined via ELISA. As shown in 2O2 caused a significant increase in the ability of cells to produce TRX. As little as 2.5 μM H2O2 caused a strong stimulation in TRX production. Maximum production was observed with 25.0–75.0 μM H2O2 whereas 100.0 μM H2O2 was inhibitory  controls , by suppressing the mRNA expression of chemokines in eosinophils after stem cell factor simulation [The present data clearly show that quercetin enhances the ability of nasal cells to produce TRX in response to stimulation with either Hmulation . Furthermulation . Synthesmulation ,32. ThesOral allergy syndrome (OAS), also recognized as pollen-food syndrome, is an allergic response in the oral cavity following the ingestion of fruits, vegetables, or nuts. OAS reportedly occurs in approximately 20–70% of patients with AR and atopy . Pollen-The results obtained from the present experiments strongly suggest that quercetin increases the ability of nasal epithelial cells, to produce TRX after stimulation with oxidants or allergens. Moreover, quercetin results in the attenuation of development of the clinical symptoms of AR by suppressing oxidative stress responses in nasal mucosa.