DCs act as sentinel cells against incoming pathogens and represent the most potent antigen presenting cells, having the unique capability to prime naïve T cells. In addition to their role in induction of adaptive immune responses, DC are also able to activate innate cells as γδ T cells; in particular, a reciprocal crosstalk between DC and γδ T cells was demonstrated. However, whether HIV infection may alter DC-Vγ9Vδ2 T cells cross-talk was not yet described. To clarify this issue, we cultured activated Vγ9Vδ2 T cells with HIV infected monocyte derived DC (MoDC). After 5 days we evaluated MoDC phenotype, and Vγ9Vδ2 T cells activation and proliferation. In our model, Vγ9Vδ2 T cells were not able to proliferate in response to HIV-infected MoDC, although an up-regulation of CD69 was observed. Upon phosphoantigens stimulation, Vγ9Vδ2 T cells proliferation and cytokine production were inhibited when cultured with HIV-infected MoDC in a cell-contact dependent way. Moreover, HIV-infected MoDC are not able to up-regulate CD86 molecules when cultured with activated Vγ9Vδ2 T cells, compared with uninfected MoDC. Further, activated Vγ9Vδ2 T cells are not able to induce HLA DR up-regulation and CCR5 down-regulation on HIV-infected MoDC. These data indicate that HIV-infected DC alter the capacity of Vγ9Vδ2 T cells to respond to their antigens, pointing out a new mechanisms of induction of Vγ9Vδ2 T cells anergy carried out by HIV, that could contribute to immune evasion. Dendritic cells (DC), characterized as the most potent antigen-presenting cells (APC), represent a multi-functional population of cells. In steady-state conditions, DC are in an immature stage and induce tolerogenic T cell responses HIV infection deeply affects several issues of immune response including DCs Studies of HIV-1 infected humans suggest that HIV infection can impact on repertoire and effector function of Vγ9Vδ2 T cells. The frequency of Vγ9Vδ2 T cells is markedly reduced in the blood of HIV-1-infected humans www.sancamilloforlanini.rm.it). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lympholyte-H . Monocytes were positively separated by anti-CD14 magnetic beads , according to manufacturer’s instructions. CD14+ cells (mean purity 95%) were then resuspended in RPMI 1640  supplemented with 10% heat-inactivated defined fetal bovine serum (FBS) (HyClone), 2 mM L-Glutammin, 10 mM HEPES buffer (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), 2 mM penicillin, and 50 µg/mL streptomycin . MoDCs were differentiated from monocytes as previously described 6/ml) were cultured for 5 days in the above described medium in the presence of 50 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng/mL interleukin-4 (IL-4) . After 5 days MoDC were infected with HIVBAL (200 ng of p24/3×106 cells) for 3 hours, then extensively washed and cultured in the above described medium (1×106/ml) (without cytokines) for 18 hours.Anonymous buffy coats of healthy donors were obtained from the Transfusion Center of San Camillo hospital . After recovering, γδ T cell viability was determined by trypan blue exclusion (>80%) and then they were co-cultured with HIV-infected MoDC  and activated with isopentenylpyrophosphate (IPP) for 5 days. In selected experiments, γδ T lymphocytes were physically separated from DC by a semi-permeable membrane . The lower compartment of the wells contained.γδ T cells were positively separated from PBMC by anti-TCR γδ microbead kit , according to manufacturer’s instructions. Purified γδ T cells (mean purity 90%) were frozen until DC differentiation. Purified γδ T cells were thawed 18 hours before activation and cultured in the above described medium (2×106 cells); the upper compartments (transwell insert) contained γδ T lymphocytes (0.5×106 cells). After 5 days, culture supernatants were collected and stored at-80°C, and cell phenotype were evaluated by flow cytometry. Where indicated γδ T cells were CFDA-SE labeled  according to manufacturer’s instructions. After 5 days, the proliferation rate was evaluated by flow cytometry.MoDC . In brief, the cells were washed twice in PBS, 1% BSA, and 0.1% sodium azide, and were stained with the mAbs for 15 min at 4°C. The cells were then washed and fixed with 4% paraformaldehyde, and analyzed using a FACS Canto II (Becton Dickinson). Since the presence of 2 purified populations, the gating strategy was performed as follow: dead cells were excluded by scatter characteristics; MoDC were identified by morphological parameters (FSC vs SSC); gated cells were then analyzed for the expression of the molecules described above. T lymphocytes were first gated by using morphological parameters; in this gate Vγ9Vδ2 T cells were identified as Vδ2+CD3+. Analysis was carried out by using Facs Diva software (Becton Dickinson). The histogram overlays were performed by FlowJo software .Cytokines released in the supernatants were analyzed by using a customized Bio-Plex Pro Assay (BIO RAD) according to manufacturer’s instructions. The assay was able to evaluate the following cytokines and chemokines: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p70), IL-13, G-CSF, IFN-γ, MCP-1, MIP-1b, RANTES, TNF-α, GM-CSF, IL-17.HIV replication was evaluated by measuring p24 released in supernatants by a microelisa system  according to manufacturer’s instructions.Results were evaluated using Mann-Whitney test. A p value <0.05 was considered statistically significant. GraphPad Prism software  was used to perform the analysis.6 MoDC), and cultured with purified γδ T cells, stained with CFDA-SE, for 5 days, and Vγ9Vδ2 proliferation was assessed by flow cytometry. 6 MoDC), and cultured with purified γδ T cells, stained with CFDA-SE, and activated with IPP at the time of co-culture. After 5 days, Vγ9Vδ2 proliferation was assessed by flow cytometry. HIV-infected MoDC strongly inhibit IPP-induced Vγ9Vδ2 T cells proliferation : CD45RA-CD27+, Effector Memory (EM): CD45RA-CD27-, Effector (EFF): CD45RA+CD27-) after culture with HIV-infected MoDC. We found that, as expected, in all conditions 60–80% of Vγ9Vδ2 T cells were CM, 10–40% EM, and the remaining fraction were naïve and EFF. Moreover, HIV-infected MoDC did not induce any alteration of the Vγ9Vδ2 T cells differentiation phenotype, in both IPP stimulated and not stimulated conditions (data not shown).Since we observed an inhibition of Vγ9Vδ2 T cells proliferation after antigen stimulation when cells are cultured with HIV-infected DC, we wondered if cytokines production was also impaired. To this end, HIV-infected MoDC were cultured with Vγ9Vδ2 T cells activated with IPP at the beginning of the co-culture, and after 5 days of culture supernatants were collected and tested for 18 cytokines. When Vγ9Vδ2 T cells were cultured with HIV-infected DC, an inhibition of IFN-γ, TNF-α and MIP1-β production was observed compared to the culture with uninfected DC , while oIt was previously shown that activated Vγ9Vδ2 T cells inhibit HIV replication in target cells, mostly by producing chemokines such as RANTES and MIP1-β Our results indicate that HIV-infected MoDC inhibit the capacity of Vγ9Vδ2 T cells to respond to IPP. Recently, it has been clearly demonstrated that butyrophilin 3A1 (BT3A1) binds phosphorylated antigens and activates Vγ9Vδ2 T cells BAL, as described above, and cultured with purified, IPP activated γδ T cells (ratio 1∶1). Five days after, we evaluated MoDC phenotype by flow cytometry. In line with the Vγ9Vδ2 T cell cytokines production involved in the DC maturation, we found that, as previously described, activated Vγ9Vδ2 T cells were able to up-regulate of CD86 on MoDC; on the contrary, when cells were HIV-infected Vγ9Vδ2 T cells failed to induce the up-regulation of CD86 , as previously shown on Vγ9Vδ2 T cells from HIV infected patients To date, it is not clear whether there exist some HIV antigens or HIV-infected cells derived antigens able to activate Vγ9Vδ2 TCR. In our model, we could not see any proliferation of Vγ9Vδ2 T cells after culture with HIV infected MoDC, nor cytokines production; however, an augmented expression of CD69 was observed. CD69 is a type II transmembrane protein and a member of the C-type lectin-like receptor family that is expressed in leukocytes upon stimulation The inhibition of T cell functions by HIV-infected DC is not limited to γδ T cells. In fact, it has been shown that HIV infection of DC alter their capacity to stimulate αβ T cells In conclusion, we show herein for the first time that MoDC infected by HIV can alter the functional capabilities of Vγ9Vδ2 T cells through a cell contact dependent mechanism. These findings provide further evidences of the complex relationship between important players of innate immunity and its modulation by HIV, that has to be take into account in evaluating new therapeutic or vaccine strategies.


Photodriven molecular motors are able to convert light energy into directional motion and hold great promise as miniaturized powering units for future nanomachines. In the current state of the art, considerable efforts have still to be made to increase the efficiency of energy transduction and devise systems that allow operation in ambient and non-damaging conditions with high rates of directional motions. The need for ultraviolet light to induce the motion of virtually all available light-driven motors especially hampers the broad applicability of these systems. We describe here a hemithioindigo-based molecular motor, which is powered exclusively by nondestructive visible light (up to 500 nm) and rotates completely directionally with kHz frequency at 20 °C. This is the fastest directional motion of a synthetic system driven by visible light to date permitting materials and biocompatible irradiation conditions to establish similarly high speeds as natural molecular motors. Although photodriven molecular motors—capable of converting light into unidirectional motion—hold promise for many applications, these typically require ultraviolet light. Here, the authors design and analyse a motor that can operate at high speeds (kHz) under visible light at ambient temperature. Thus, our new molecular motor system enables for the first time to power fast unidirectional rotation under ambient and non-damaging conditions, representing the next crucial step towards developing highly efficient nanomachines that work at materials and biocompatible conditions.Synthetic photo-driven molecular motors123468910Z/E or E/Z photoisomerization343536R- or S-configuration), and sterical crowding at the ring-fused stilbene fragment led to helical twisting (P- or M-helicity) around the central double bond. Combined with its Z and E isomeric forms, motor 1 can thus assume four different diastereomeric structures for each configuration of the stereogenic sulfur centre, for example, Z-(S)-(P), Z-(S)-(M), E-(S)-(M) and E-(S)-(P), which can be distinguished by conventional spectroscopic methods. This molecular set-up is related to the Feringa motor system consisting of a sterically overcrowded double bond in combination with carbon-based stereogenic centres1Our new molecular motor is based on the hemithioindigo (HTI) chromophore262728293031321 was synthesized in three steps starting from known, easily accessible precursors  was methylated twice in the 2-position using sodium hydride and methyl iodide to give indanone 5. Benzothiophenone 3 was subsequently condensed with indanone 5 using boron trifluoride diethyl etherate, after which a final oxidation with hydrogen peroxide furnished sulfoxide motor 1. Crystals suitable for X-ray analysis were obtained for two isomeric forms of racemic motor 1, the Z-(S)-(P)/Z-(R)-(M) (CCDC 1061969) and the E-(S)-(P)/E-(R)-(M) (CCDC 1061970) isomers, enantiomerically pure Z-(R)-(M) (CCDC 1406625) isomer  isomer , as wellS) and (R) enantiomeric forms of motor 1 are stable -(P)/Z-(R)-(M) isomers are the thermodynamically stable form of motor 1, which can be obtained in 75% yield by heating a toluene-d8 solution of 1 to 100 °C for 8 h, as analysed by nuclear magnetic resonance (NMR) spectroscopy. Under these conditions also 25% of the E-(S)-(P)/E-(R)-(M) isomers are obtained. When a solution of 1 in xylene-d10 is heated to 130 °C for 12 h, 73% of the Z-(S)-(P)/Z-(R)-(M) isomers and 27% of the E-(S)-(P)/E-(R)-(M) isomers are obtained -(P)/E-(R)-(M) to the Z-(S)-(P)/Z-(R)-(M) isomers provided an energy barrier of 29.54 kcal mol−1 for the process (E-(S)-(P)/E-(R)-(M) isomers at 25 °C, the two Z and E diastereomers could conveniently be separated by conventional chromatography and studied individually. Direct assignment of the two separated species to the corresponding diastereomers was straightforward in solution, as NOESY NMR spectra showed specific cross-signals between the thioindigo and the stilbene part of the molecule (Z-(S)-(P)/Z-(R)-(M) isomers (CCDC 1061969). The 1H NMR and ultraviolet/visible (ultraviolet/vis) spectra of the same crystal batch were recorded, showing only one single species to be present in solution. Thus, we could directly assign the corresponding NMR signals as well as the ultraviolet/vis spectrum to the Z-(S)-(P)/Z-(R)-(M) isomers -(P)/E-(R)-(M) isomeric form (see 1 (Z (up to 470 nm) and bathochromic absorption of the E isomer (up to 505 nm)—is similar to typical HTI photoswitchesIn a similar way, the first fraction obtained after chromatographic separation and corresponding NMR and ultraviolet/vis solution spectra could be assigned to the m (see 1 —hypsochrZ-(S)-(P) and Z-(R)-(M) isomers (CCDC 1061969) represent the global thermodynamic minimum of motor 1; the structure of the Z-(S)-(P) isomer is shown in Z-(R)-(M) isomer -(P)/Z-(R)-(M)-1 in solution is very similar to the one found in the crystal.In solution, less structural information is available but several details could be inferred from NMR analysis. The NOESY NMR spectrum  allowed spectrum  establis1, the E-(S)-(P)/E-(R)-(M) isomers represent the most stable configuration. The crystal structure of the E-(S)-(P) isomer (CCDC 1061970) is shown in Among the metastable forms of motor Z-(S)-(P)/Z-(R)-(M) isomers (E-(S)-(P)/E-(R)-(M)-1 and the one found in the crystal.An NMR analysis similar to the one conducted for the  isomers  showed a1 in CD2Cl2 with 405 nm light at 23 °C, a photostationary state (PSS) consisting of 22% Z-(S)-(P)/Z-(R)-(M) and 78% E-(S)-(P)/E-(R)-(M) isomers is formed, as judged by integration of indicative signals in the 1H NMR spectrum. Irradiation with 490 nm light leads to a PSS of 83% Z-(S)-(P)/Z-(R)-(M) and 17% E-(S)-(P)/E-(R)-(M) isomers. The quantum yields for photoisomerization at ambient temperature are 30% for the Z to E isomerization and 12% for the E to Z isomerization. Thus, at 23 °C only two different diastereomeric forms are observed under irradiation conditions (1H NMR spectrum of Z-(S)-(P)/Z-(R)-(M)-1 did not change significantly, which preserved signal identity (Z-(S)-(P)/Z-(R)-(M)-1 in CD2Cl2 was irradiated with 460 nm light at –90 °C, a new set of signals was observed in up to 45% signal intensity. (E-(S)-(P)/E-(R)-(M) isomers than to those of the Z-(S)-(P)/Z-(R)-(M) isomers. Switching off irradiation leads to the decrease of the new set of signals and at the same time to a similar increase of only the E-(S)-(P)/E-(R)-(M) isomer signals. After 1 h, the new set of signals had completely disappeared. An additional annealing experiment also demonstrated the complete thermal conversion of these signals to the E-(S)-(P)/E-(R)-(M) isomer signals (Z-(S)-(P)/Z-(R)-(M)-1 leads exclusively to the population of an intermediate E isomeric state before a thermal step converts it completely (>95%) into the E-(S)-(P)/E-(R)-(M) isomers. We therefore assign the new set of signals to the E-(S)-(M)/E-(R)-(P) isomeric form. An energy barrier ΔG* of 13.1 kcal mol−1 was found for the thermal conversion between the two E isomers by first-order kinetic analysis (E-(S)-(M)/E-(R)-(P) isomeric state is populated at higher temperatures, a CD2Cl2 solution of E-(S)-(P)/E-(R)-(M)-1 at 22 °C was shock-frozen in liquid N2 and allowed to warm to –90 °C to record a 1H NMR spectrum at this temperature immediately after thawing. No signals of the E-(S)-(M)/E-(R)-(P) isomers were observed in this experiment, showing that also at 22 °C the E-(S)-(P)/E-(R)-(M) isomers are present in >95% -(P)/E-(R)-(M)-1 at –90 °C (Z-(S)-(P)/Z-(R)-(M)-1 were found to build up immediately after irradiation. At higher power of irradiation also the E-(S)-(M)/E-(R)-(P) isomers were observed, but only as a photoproduct of the Z-(S)-(P)/Z-(R)-(M) isomers after the latter were already present -(M)/Z-(R)-(P) isomers were not found at the NMR-accessible temperature range. The barrier for the thermal conversion of Z-(S)-(M)/Z-(R)-(P)-1 to Z-(S)-(P)/Z-(R)-(M)-1 must therefore be <12.0 kcal mol−1.Complementary experiments were performed with a CDt –90 °C , using a present . An irraric form . Thus, tZ-(S)-(M)/Z-(R)-(P) intermediate, our experiments established that under the same irradiation conditions (460 nm) the Z-(S)-(P)/Z-(R)-(M) and E-(S)-(P)/E-(R)-(M) isomers are interconverted via two different pathways. Z-(S)-(P)/Z-(R)-(M)-1 is first converted photochemically into the E-(S)-(M)/E-(R)-(P) isomers. From this intermediate state a thermal step leads to complete formation of E-(S)-(P)/E-(R)-(M)-1, which breaks microscopic reversibility. In contrast, photoirradiation of E-(S)-(P)/E-(R)-(M)-1 leads to Z-(S)-(P)/Z-(R)-(M)-1, omitting the E-(S)-(M)/E-(R)-(P) isomeric state, which otherwise would have been observable in our experiments. Since each conversion constitutes a 180° rotation around the central double bond, the observed two different pathways are only consistent with a unidirectional 360° rotation. As the thermal step converting the E isomeric forms proceeds to >95% and photoirradiation of the E-(S)-(P)/E-(R)-(M) isomers did not populate any of the intermediate E-(S)-(M)/E-(R)-(P) state, we conclude that 1 rotates with >95% in one direction. This finding gives indirect evidence that an intermediate Z-(S)-(M)/Z-(R)-(P) isomeric state must exist, which is at least 1.1 kcal mol−1 higher in energy than the Z-(S)-(P)/Z-(R)-(M) state.Although we could not directly observe the 1 is the thermal E-(S)-(M)/E-(R)-(P) to E-(S)-(P)/E-(R)-(M) conversion. With the established energy barrier of 13.1 kcal mol−1, a maximum rotation frequency at 20 °C of 1 kHz can be realized, which is the fastest rate for a molecular motor powered by visible light to date. Photoisomerization of the Z-(S)-(P)/Z-(R)-(M) isomers with a 505 nm diode also resulted in the formation of E-(S)-(P)/E-(R)-(M) isomers (1 can be operated at a wide range of wavelengths in the visible part of the spectrum. Separate irradiation of both pure Z-(S)-(P)/Z-(R)-(M) and E-(S)-(P)/E-(R)-(M) isomers with direct sunlight for 20 min resulted in a photostationary state composed of 47% of the former and 53% of the latter isomers showing that motor 1 can efficiently be powered using sunlight -(P) and E-(S)-(P) isomers were optimized starting from the crystal analytical data of the Z-(S)-(P) isomer. The optimized geometries and the geometries obtained experimentally from crystal structure analysis agree very well (see Z-(S)-(M) and E-(S)-(M) isomers were optimized starting from the helical inverted structures of Z-(S)-(P) and E-(S)-(P) isomers, which were then allowed to fully relax. Transition states were then obtained starting from the partially restricted Z-(S)-(M) and E-(S)-(M) minimum structures, which were optimized fully relaxed at a second stage of the calculations (Z-(S)-(P) isomer is found indeed as a global minimum structure in the theoretical description, while the E-(S)-(P) isomer is only 0.8 kcal mol−1 higher in energy. This finding is perfectly confirmed experimentally by the 75:25 and 73:27 ratio of Z-(S)-(P) and E-(S)-(P) isomers observed in solution after heating to 100 °C and 130 °C, respectively . The E-(S)-(M) isomer is 3.03 kcal mol−1 higher in energy compared with the E-(S)-(P) isomer in the theoretical description, which explains why the E-(S)-(M) isomer is completely converted  to the E-(S)-(P) isomer in the thermal step. For this conversion, the theoretical description gives a barrier of 14.62 kcal mol−1, a value that again agrees remarkably well with the experimentally measured 13.10 kcal mol−1. Interestingly, the calculated energy barrier of the corresponding thermal conversion of Z-(S)-(M) to Z-(S)-(P) is considerably smaller with only 5.54 kcal mol−1. Thus, a half-life of 0.75 μs would be found for this process at –90 °C—a time range that is not accessible with conventional low-temperature NMR or ultraviolet/vis spectroscopic techniques. The theoretically found energy difference between the Z-(S)-(M) and Z-(S)-(P) isomers is 3.26 kcal mol−1, which is sufficiently high to convert the Z-(S)-(M) isomer completely in the thermal step. Again, this is in good agreement with our experiments, as we observe only Z-(S)-(P)/Z-(R)-(M) to E-(S)-(M)/E-(R)-(P) and not Z-(S)-(P)/Z-(R)-(M) to E-(S)-(P)/E-(R)-(M) photoisomerization when irradiating the Z-(S)-(P)/Z-(R)-(M) isomers of motor 1.To gain deeper insight into the function of our new motor system, we performed a theoretical analysis of the ground-state energy profile at the DFT MPW1K level of theory using the 6–31+G basis set . The strulations . All opt1H NMR chemical shifts at the DFT MPW1K level of theory using the 6–31+G basis set. A detailed comparison of the chemical shifts obtained experimentally at –90 °C in CD2Cl2 solution and the theoretical values is given in 1 are perfectly reproduced in the theoretical description, which further supports our assignment of the thermally unstable intermediate to the E-(S)-(M)/E-(R)-(P) isomers of motor 1. In addition, the extinction coefficients and circular dichroism spectra of all four diastereomers were calculated using the same level of theory but an increased 6–311++G basis set -(P), Z-(R)-(M), E-(S)-(P) and E-(R)-(M), was possible in one single run using a CHIRALPAK IC column from Diacel. Circular dichroism spectra of these four isomers were measured (Z-(R)-(M) isomer  with >95% unidirectionality, which sets a new standard to the performance and sustainability of synthetic molecular motors. With these exceptional properties, motor 1 can be used as a highly efficient molecular power unit for applications in sensitive environments that do not tolerate ultraviolet light or high temperatures. We believe that especially in the fields of material sciences and biology, our new motor system will be of great advantage and we will direct our future efforts towards the implementation of motor 1 into more complex nanomachinery and biological systems.We have developed a novel HTI-based molecular motor 2 60  and distilled technical solvents. HPLC was performed on a Shimadzu HPLC system consisting of an LC-20AP solvent delivery module, a CTO-20A column oven, a SPD-M20A photodiode array ultraviolet/vis detector and a CBM-20A system controller using a semipreparative CHIRALPAK IC column (particle size 5 μm) from Diacel and HPLC grade solvents (2-PrOH and n-heptane) from Sigma-Aldrich and ROTH. 1H NMR and 13C NMR spectra were measured on a JEOL ECX 400 (400 MHz), Varian VNMRS 400 (400 MHz), Varian VNMRS 600 (600 MHz) or Bruker AVANCE III HD 800 (800 MHz) NMR spectrometer. Chemical shifts (δ) are reported in p.p.m. relative to the signal of tetramethylsilane. Residual solvent signals in the 1H and 13C NMR spectra were used as internal reference . The resonance multiplicity is indicated as s (singlet), d (doublet) or t (triplet). The coupling constant values (J) are given in hertz (Hz). Electron impact mass spectra were measured on a Finnigan MAT95 mass spectrometer. The most important signals are reported in m/z units with M as the molecular ion. Elemental analyses were performed in the micro analytical laboratory of the LMU Department of Chemistry on an Elementar Vario EL apparatus. Infrared spectra were recorded on a Perkin Elmer Spectrum BX-FT-IR instrument equipped with a Smith DuraSamplIR II ATR-device. Transmittance values are qualitatively described by wavenumber (cm–1) as very strong (vs), strong (s), medium (m), weak (w) and very weak (vw). Ultraviolet/vis spectra were measured on a Varian Cary 50 spectrophotometer. The spectra were recorded in CH2Cl2 in a quartz cuvette (1 cm). Absorption wavelengths (λ) are reported in nm and the extinction coefficients (ɛ) in l mol–1 cm–1. Melting points (m.p.) were measured on a Büchi B-540 melting point apparatus in open capillaries.Reagents and solvents were obtained from Acros, Aldrich, Fluka, Merck or Sigma-Aldrich in the qualities puriss., p.a. or purum and used as received unless stated otherwise. Technical solvents were distilled before use for column chromatography and extraction on a rotary evaporator . Thin-layer chromatography (TLC) was conducted on Merck Silica 60 F254 TLC plates and visualization conducted with a ultraviolet lamp (254 or 366 nm). Deuterated solvents were obtained from Cambridge Isotope Laboratories and were used without further purification. Thiophenol and 4,7-dimethoxy-1-indanone were purchased at reagent grade from Sigma-Aldrich and were used as received. Boron trifluoride diethyl etherate was purchased from ABCR and freshly distilled before use. Column chromatography was performed with SiO2Cl2, diethyl ether-d10, toluene-d8 or o-xylene-d10). Irradiations at 23 °C were conducted using light-emitting diodes (LEDs) from Roithner Lasertechnik GmbH . For low-temperature studies a Prizmatix Mic-LED-365 high-power collimated LED (365 nm), a Prizmatix Mic-LED-415 high-power collimated LED (415 nm) or a Prizmatix UHP-Mic-LED-460 ultra high-power collimated LED (460 nm) was used as light source, and the light beam was guided by a fibre-optic cable  and pointed directly into the NMR tube during NMR measurements. The quantum yields of Z-(S)-(P)/Z-(R)-(M) to E-(S)-(P)/E-(R)-(M) photoisomerization and vice versa were measured at 22 °C in CH2Cl2. A solution of the respective isomer was irradiated at 420 nm for a certain time  and the absorption changes were recorded. Using the Beer–Lambert law at suitable wavelengths the molar changes could be calculated. The amounts of absorbed photons were determined using 2-(4-methoxybenzylidene)-1-benzothiophen-3(2H)-one as calibration standard with known photoisomerization quantum yieldZ-(S)-(P)/Z-(R)-(M) or E-(S)-(P)/E-(R)-(M) isomers was used in the measurements to ensure equal amounts of absorbed photons in each case.Continuous irradiation of the solutions was conducted in NMR tubes in different deuterated solvents (CD2) was prepared according to a literature procedure5) was prepared from a commercially available 4,7-dimethoxy-1-indanone based on an altered literature procedure2-Phenylthioacetic acid (2Cl2 (3 × 150 ml), the combined organic phases were dried over sodium sulfate and the solvent was removed in vacuo. For the following synthetic step, crude benzothiophenone 3 was used without further purification.To 2-phenylthioacetic acid , thionylchloride  and dimethyl formamide (3 drops) were added, and the solution was stirred under reflux for 1 h. After evaporation of residual thionylchloride at 50 °C, the remaining 2-phenylthioacetic acid chloride was dissolved in 1,2-dichloroethane (5 ml) and subsequently cooled to 0 °C. Aluminium chloride  was added slowly over a period of 2 min, followed by stirring for 5 min at 0 °C. The reaction mixture was allowed to warm to 23 °C and, after stirring for 1 h at this temperature, was poured on ice/water (150 ml). The aqueous phase was extracted with CH1H-inden-1-one  in glyme (5 ml) was added under argon atmosphere to a solution of sodium hydride  in glyme (2 ml). After stirring the resulting solution under reflux for 45 min, the mixture was cooled to 0 °C and methyl iodide  was added dropwise followed by stirring for 24 h at 23 °C. After addition of a saturated aqueous solution of ammonium chloride (100 ml), the mixture was extracted with ethyl acetate (3 × 150 ml). The combined organic phases were dried over sodium sulfate and the volatiles were removed in vacuo. The crude product was purified by flash column chromatography  to afford indanone 5  as a colourless liquid. TLC : Rf=0.15; 1H-NMR : δ 6.98 =8.7 Hz, 1H, H-C(2)), 6.73 =8.7 Hz 1H, H-C(1)), 3.89 ), 3.84 ), 2.85 ), 1.21 ), H-C(14)); 13C-NMR : δ 209.6 (C(9)), 152.4 (C(6)), 150.7 (C(3)), 143.1 (C(4)), 124.7 (C(5)), 116.9 (C(2)), 109.7 (C(1)), 56.2 (C(15), C(16)), 45.8 (C(8)), 39.5 (C(7)), 25.8 (C(13), C(14)); high-resolution electron ionization-MS (HREI-MS) (m/z): [M]+ calcd. for C13H16O, 220.1099; found, 220.1089.A solution of 4,7-dimethoxy-2,3-dihydro-3  and 5  in CH2Cl2 (2.50 ml) boron trifluoride diethyl etherate  was added and stirred for 24 h at 23 °C. Boron trifluoride diethyl etherate  was added again and the resulting solution was stirred for another 24 h at 23 °C. The reaction mixture was then quenched with water (50 ml), extracted with ethyl acetate (3 × 50 ml) and dried over sodium sulfate. The solvent was removed in vacuo and the crude product was dissolved in a mixture of acetic acid (14 ml) and hydrogen peroxide . After 2 h of stirring at 23 °C, the reaction mixture was neutralized with a saturated aqueous solution of sodium bicarbonate (150 ml). After extraction with ethyl acetate (3 × 150 ml), the combined organic layers were dried over sodium sulfate and the solvent was removed in vacuo. The crude mixture was purified by flash column chromatography  to afford motor 1  as a yellow solid.To a solution of Z-(S)-(P)/Z-(R)-(M) isomers: m.p: 215 °C; TLC : Rf=0.08; 1H NMR : δ 7.99 =7.7 Hz, 1H, H-C(6)), 7.95 =7.7 Hz, 1H, H-C(3)), 7.83 =7.5, 1.2 Hz, 1H, H-C(5)), 7.69 =7.4, 1.0 Hz, 1H, H-C(4)), 7.05 =8.8 Hz, 1H, H-C(14)), 6.85 =8.8 Hz, 1H, H-C(13)), 4.03 ), 3.83 ), 2.99 =16.4 Hz, 1H, H-C(18)), 2.91 =16.4 Hz, 1H, H-C(18′)), 1.54 ), 1.52 ); 13C-NMR : δ 185.4 (C(1)), 170.7 (C(9)), 152.0 (C(11)), 151.1 (C(7)), 150.6 (C(15)), 144.0 (C(8)), 139.3 (C(17)), 136.3 (C(2)), 135.6 (C(5)), 132.2 (C(4)), 127.0 (C(10)), 126.8 (C(6)), 125.0 (C(3)), 116.9 (C(14)), 110.0 (C(13)), 56.5 (C(16)), 55.5 (C(12)), 52.4 (C(19)), 47.6 (C(18)), 28.6 (C(20)), 25.7 (C(21)); IR : 3425w, 2940w, 2663w, 2476vw, 2004vw, 1673s, 1586m, 1541s, 1488s, 1451s, 1381w, 1360w, 1334m, 1319w, 1292m, 1194m, 1171m, 1113m, 1099m, 1063vs, 1035vs, 1004m, 991m, 955s, 938m, 916m, 879m, 821m, 806s, 790m, 759s, 748s, 713s, 696m, 669m, 689m, 660s cm–1; ultraviolet/vis (CH2Cl2): λmax (ɛ) 400 , 355 , 341  nm; HREI-MS (m/z): [M]+ calcd. C21H20O4S, 368.1082; found, 368.1070; analysis : C , H , S .E-(S)-(P)/E-(R)-(M) isomers: m.p: 186 °C; TLC : Rf=0.26; 1H NMR : δ 8.06 =7.7 Hz, 1H, H-C(6)), 7.98 =7.5 Hz, 1H, H-C(3)), 7.85 =7.5, 1.3 Hz, 1H, H-C(5)), 7.73 =7.4, 1.0 Hz, 1H, H-C(4)), 6.99 =8.9 Hz, 1H, H-C(14)), 6.76 =8.8 Hz, 1H, H-C(13)), 3.84 ), 3.76 ), 3.07 =15.4 Hz, 1H, H-C(18)), 2.90 =15.4 Hz, 1H, H-C(18′)), 1.95 ), 1.38 ); 13C-NMR : δ 182.7 (C(1)), 169.2 (C(9)), 153.0 (C(11)), 149.8 (C(15)), 149.2 (C(7)), 140.0 (C(8)), 137.4 (C(17)), 135.5 (C(2)), 134.7 (C(5)), 132.3 (C(4)), 128.7 (C(10)), 126.8 (C(6)), 124.8 (C(3)), 115.7 (C(14)), 109.6 (C(13)), 56.0 (C(16)), 55.1 (C(12)), 52.3 (C(19)), 46.3 (C(18)), 28.1 (C(21)), 25.8 (C(20)); IR : 3419m, 2954m, 2834vw, 2780vw, 2666w, 2568vw, 2512vw, 2471vw, 2155vw, 1675vs, 1588m, 1548m, 1491s, 1465s, 1451s, 1437m, 1412m, 1384m, 1363w, 1334m, 1317m, 1262s, 1215s, 1172m, 1163m, 1137w, 1118m, 1097m, 1070s, 1037s, 1002s, 959s, 863m, 797m, 783m, 753vs, 711s, 668s, 668vs cm–1; ultraviolet/vis (CH2Cl2): λmax (ɛ) 420 , 341  nm. For further experimental details and characterization data, see 1 are given in the Z-(S)-(P)/Z-(R)-(M) and E-(S)-(P)/E-(R)-(M) isomers found in the corresponding crystal structures are compared to the theoretically obtained geometries in The details of the theoretical description of motor Accession codes: The X-ray crystallographic coordinates for structures reported in this study have been deposited at the Cambridge Crystallographic Data Centre (CCDC), under CCDC numbers 1061969, 1061970, 1406625, 1408096 and 1408097. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.How to cite this article: Guentner, M. et al. Sunlight-powered kHz rotation of a hemithioindigo-based molecular motor. Nat. Commun. 6:8406 doi: 10.1038/ncomms9406 (2015).Supplementary Figures 1-41, Supplementary Tables 1-4, Supplementary Methods and Supplementary References.MPW1K_Minimum_ESM_Isomer.cif = minimum structure of the E-(S)-(M) isomer of motor 1 calculated at the MPW1K level of theory using a 6-31+G basis setMPW1K_Minimum_ESP_Isomer.cif = minimum structure of the E-(S)-(P) isomer of motor 1 calculated at the MPW1K level of theory using a 6-31+G basis setMPW1K_Minimum_ZSM_Isomer.cif = minimum structure of the Z-(S)-(M) isomer of motor 1 calculated at the MPW1K level of theory using a 6-31+G basis setMPW1K_Minimum_ZSP_Isomer.cif = minimum structure of the Z-(S)-(P) isomer of motor 1 calculated at the MPW1K level of theory using a 6-31+G basis setMPW1K_TransitionState_ESM_ESP.cif = transition state structure of the conversion from E-(S)-(M) to E-(S)-(P) isomer of motor 1 calculated at the MPW1K level of theory using a 6-31+G basis setMPW1K_TransitionState_ZSM_ZSP.cif = transition state structure of the conversion from Z-(S)-(M) to Z-(S)-(P) isomer of motor 1 calculated at the MPW1K level of theory using a 6-31+G basis set


In the crystal, adducts are linked by C—H⋯O and C—H⋯Br hydrogen bonds, forming a two-dimensional network.The first crystal structure determination of a 1:2 co-crystalline adduct of 1,3,6,8-tetraazatricyclododecane (TATD) and 4-bromophenol, has been determined. The asymmetric unit of the title co-crystalline adduct comprises a half molecule of aminal cage polyamine plus a 4-bromophenol molecule. A twofold rotation axis generates the other half of the adduct. The primary inter-species association in the title compound is through two intermolecular O—H⋯N hydrogen bonds. In the crystal, the adducts are linked by weak non-conventional C—H⋯O and C—H⋯Br hydrogen bonds, giving a two-dimensional supramolecular structure parallel to the bc plane.The structure of the 1:2 co-crystalline adduct C Unlike al., 2007, where aet al., 2007aK values between the species involved in the hydrogen bond , p-bromophenol is more acidic (paK = 9.37)  Å; O—H⋯N 158 (7)°)] in the title compound are compared to the values found in the TATD:hydroquinone, 1:1 adduct   and Br1—C4 1.907 (5) Å.A database search  for 4-bromophenol yielded 17 hits with 21 fragments. The mean C—O bond length in these structures is 1.35 (5) Å and the mean C—Br bond length is 1.91 (3) Å. These values are in excellent agreement with those of the title compound, 3,8]dodecane yielded only three hits, two determinations of the compound itself   and 4-bromophenol  were manually mixed in a mortar with pestle at room temperature for 20 min as required to complete the reaction (TLC). The mixture was then dissolved in a minimum amount of methanol and left to crystallize at room temperature. Subsequent recrystallization with MeOH then yielded the title compound as white crystals in 78% yield, m.p. = 367–368 K.1,3,6,8-tetraazatricyclo[4.4.1.1Uiso(H) set to 1.2Ueq of the parent atom.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015006684/sj5449sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989015006684/sj5449Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015006684/sj5449Isup3.cmlSupporting information file. DOI: 1057775CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Engleromyces goetzii is a traditional medicinal mushroom that is widely used to treat infection, inflammation and cancer in Tibet, Sichuan and Yunnan provinces of China. Two new trichothecenes, engleromycones A and B (1 and 2), one new cuparane-type sesquiterpenoid named infuscol F (11), eight known trichothecene analogs, sambucinol (3), 3-deoxysambucinol (4), trichothecolone (5), trichodermol (6), 8-deoxytrichothecin (7), trichothecin (8), trichothecinol B (9) and trichothecinol A (10), and one known cyclopentanoid sesquiterpene cyclonerodiol (12) were isolated from the cultures of E. goetzii. The new compounds were elucidated through spectroscopic analyses. The anticancer effects of trichothecenes 1–10 were examined in the HL-60, SMMC-7721, A549, MCF-7, and SW-480 human cancer cell lines using an MTT assay. Trichothecinol A (10) significantly inhibited the growth of MCF-7 cells, with an IC50 value of 0.006 µM, which was comparable to the cytotoxic activity of the positive control, paclitaxel, indicating that trichothecinol A (10) represents a potential anticancer agent.The online version of this article (doi:10.1007/s13659-014-0051-1) contains supplementary material, which is available to authorized users. Ganoderma lucidum, Hericium erinaceus and Antrodia cinnamomea, have historically been used in Asia for medicinal purposes + ion at m/z 371.1472 in positive HR ESI MS analysis, corresponding to the molecular formula C19H24O6 with 8° of unsaturation. The 1H NMR spectrum of 1 exhibited four methyl groups at δH 0.91 , 1.08 , 1.84  and 2.14  and three olefinic protons at δH 5.74  and 6.40 . A careful analysis of the 1H NMR signals at δH 5.74 , 6.40 , and 2.14  and the 13C NMR (DEPT) signals at δC 165.2 (s), 119.7 (d), 146.9 (d), and 15.4 (q) revealed a –OCOCH=CHCH3 substituent group. The remaining NMR signals indicated three methyl groups, three methylenes, three methines and six quaternary carbons, suggesting that 1 was a sesquiterpene. The 1H and 13C NMR spectral data of 1 were similar to those of loukacinal A +; 349 (60) [M + H]+; 719 (15) [2M + Na]+; HR ESI MS (positive) m/z 371.1,472 [M + Na]+ .Colorless oil; mentclass2pt{minimc 0.2, MeOH); UV (MeOH): λmax (log ε) 211 (3.46) nm; IR (KBr): νmax 2984, 2940, 2926, 1713, 1650, 1437, 1415, 1181, 1026 cm−1; 1H and 13C NMR data see Table m/z 339 (30) [M + Na]+; 655 (10) [2M + Na]+; HR ESI MS (positive) m/z 339.1563 [M + Na]+ .Colorless oil; mentclass2pt{minimc 0.08, MeOH); UV (MeOH): λmax (log ε) 203 (3.97) nm; IR (KBr): νmax 3441, 3265, 2957, 2933, 2875, 1640, 1413 cm−1; 1H and 13C NMR data see Table m/z 261 (100) [M + Na]+; 499 (50) [2M + Na]+; HR ESI MS (positive) m/z 261.1825 [M + Na]+ .Colorless oil; 2. The cytotoxicity assay was performed according to the MTT -2,5-diphenyltetrazolium bromide) method in 96-well microplates. Briefly, 100 µL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h prior to drug addition, whereas the suspended cells were seeded immediately prior to drug addition, both with an initial density of 1 × 105 cells/mL. Each tumor cell line was exposed to the test compounds at various concentrations in triplicate for 48 h, with cisplatin and paclitaxel as the positive controls. Following incubation, MTT (100 µg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 200 µL of SDS following the removal of 100 µL of the medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated using Reed and Muench’s method [The human tumor cell lines HL-60 (promyelocytic leukemia), SMMC-7712 (hepatocarcinoma), A549 (lung carcinoma), MCF-7 (breast adenocarcinoma), SW-480 (colon adenocarcinoma) and BEAS-2B normal cell line  were used. All of the cells were cultured in RMPI-1640 or DMEM medium  plus 10 % fetal bovine serum (HyClone) at 37 °C in a humidified atmosphere with 5 % COs method .Supplementary material 1 (PDF 1496 kb)Below is the link to the electronic supplementary material.


In the crystals of both compounds, molecules are linked via bifurcated C—H⋯ hydrogen bonds, forming chains propagating along [100] and along [010].The title compounds were synthesized  20H16ClO2P, (I), and C18H21O2P, (II), were synthesized via an Arbuzov reaction between an α-bromoketone and isopropoxydiphenylphosphane. In the crystals of both compounds, molecules are linked via bifurcated C—H⋯ hydrogen bonds, forming chains propagating along [100] for (I) and along [010] for (II). The chains are linked via C—H⋯π interactions, leading to the formation of sheets lying parallel to (010) for (I) and (001) for (II). The absolute structure of compound (II) was determined by resonant scattering [Flack parameter = 0.088 (14)].The title compounds, C Direct excitation of lanthanides is difficult due to the parity forbidden f–f transitions required and relatively low molar absorptivities, but fortunately this excitation can be sensitized with an appropriate organic ligand. The ligand acts as an antenna by harvesting the excitation energy and transferring this energy to the metal emitting state  group has been shown to be an effective ligand for the sensitization of lanthanide luminescence P21. In compound (I)anti to one another, most probably to minimize unfavourable dipole–dipole interactions. In compound (II)syn to one another.The molecular structures of compounds (I)a axis, and as the individual molecules stack in this direction they appear to rotate around the chlorine atom that lies close to the twofold screw axis, creating a pinwheel arrangement of molecules  hydrogen bonds, forming chains propagating along [100]; see Fig. 3via C—H⋯π interactions es Fig. 3. The mols Table 1, formingb axis), but in this case, the P1—C2 bond in the center of the molecule lies about a twofold screw axis and acts as the pivot point for the pinwheel arrangement rather than the terminal chlorine atom as seen in the crystal of compound (I)via bifurcated C—H⋯ hydrogen bonds, forming chains propagating along [010] (see Table 2via C—H⋯π interactions (Table 2Compound (II)s Table 2, formingviz. E--3,6-dimethyl-5-diphenylphosphinoyl-7-triphenylmethoxyhept-2-en-4-one acetone solvate -2--4-diphenylphosphinoyl-1-phenylpentan-3-one monohydrate -4,5-dihydroxy-1,5-diphenyl-2-(diphenylphosphinoyl)pentan-1-one) , as does compound (II)ca 67.97, 73.25 and 68.24°, respectively, similar to the arrangement in compound (I)The Cambridge Structural Database Uiso(H)= 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015006994/su5111sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989015006994/su5111Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015006994/su5111IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989015006994/su5111Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015006994/su5111IIsup5.cmlSupporting information file. DOI: 1058397, 1012828CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Ternary complex (TC) and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway, respectively. How TC and eIF4F assembly are coordinated, however, remains largely unknown. We show that mTOR suppresses translation of mRNAs activated under short-term stress wherein TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2β phosphorylation and recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2β mediates the effect of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2β and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation, whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation. Ternary complex (TC) and eIF4F complex assembly are rate-limiting steps in translation initiation that are regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway. Here the authors show that the protein kinases mTORC1 and CK2 coordinate TC and eIF4F complex assembly through eIF2β to stimulate cell proliferation. Messenger RNA (mRNA) translation plays a major role in homeostasis, whereas its dysregulation underpins a variety of pathological states including cancer, metabolic syndrome and neurological disordersMet ref. . MammaliiMet, eIF2-bound GTP is hydrolyzed to GDP and the TC complex is recycled by the guanine nucleotide exchange factor (GEF) eIF2B, which converts eIF2:GDP to eIF2:GTP1eIF2 is a heterotrimer that comprises eIF2α, β and γ subunitsP<5.2e−12). In turn, addition of the active-site mTOR inhibitor torin1 (ref. P<3.7e−16). These effects of modulation of mTOR signalling on translation of ‘eIF2α-sensitive' mRNAs were not caused by the changes in cytoplasmic mRNA levels  was determinedin1 ref.  to insulA levels .Insulin induced mTORC1 signalling as illustrated by elevated phosphorylation of 4E-BPs and the S6 kinase (S6K) substrate ribosomal protein S6 (rpS6) as compared with control serum-starved cells, which was reverted by the allosteric mTOR inhibitor rapamycin or active-site mTOR inhibitor (KU-0063794 and torin1) 910. TherefoIt has been reported that Casein kinase 2 (CK2), which is stimulated by insulin and serum13+/+ and PTEN−/− cells+/+ cells, CX-4945 strongly reduced PTEN phosphorylation and mTOR signalling as judged by reduction in rpS6 and 4E-BP1 phosphorylation  wherein mTOR is activated by the loss of a negative upstream regulator TSC1/2 , when Ser2 and Ser67 were substituted with alanines A), or when TOS motif was disrupted   . Reduced mutant) . CK2 wasse assay . This exmulation . Disruptmulation , but notmulation . Notablymulation . Taken tmulation . Either mulation , thus in35S-labelling D eIF2β mutant D eIF2β-expressing cells  and phosphatases )We next investigated whether eIF2β mediates effects of mTORC1 on eIF2α phosphorylation and mRNA translation. To avoid interference of the endogenous eIF2β, and since depletion of eIF2β reduced cell viabilityβ mutant . In turnng cells . eIF2α png cells  by immunng cells . In turnng cells , therebyin vitro binding assay+/+ cells, whereas 1-h exposure to CX-4945 was insufficient to decrease NCK1:WT eIF2β association and WT eIF2β phosphorylation in HCT116 PTEN−/− cells , which has been implicated in recruitment of PP1 to eIF2 leading to eIF2α dephosphorylation/− cells . In turn/− cells . These fOur results indicate that eIF2β phosphorylation mediates the effects of mTORC1 and CK2 on TC formation and global protein synthesis. Since global protein synthesis rates closely correlate with proliferation ratesiMet 6061Our results show that eIF2β phosphorylation-dependent stimulation of TC formation is synchronized with eIF4F complex assembly, which is mediated by CK2 and mTORC1. Inactivation of 4E-BPs by mTORC1-dependent phosphorylation stimulates eIF4F complex assemblyL-glutamine . HCT116 PTEN+/+ and PTEN−/− cells were obtained from Dr WaldmanL-glutamine. U2OS cells that overexpress CK2α/β under the control of TetOFF systemL-glutamine and 1.5 μg ml−1 tetracycline (Sigma-Aldrich) to suppress CK2α/β expression. CK2α/β expression was induced by washing the cells three times in 1 × PBS, and incubating them in tetracycline-free media for 16 h before treating cells as indicated in figure legends. HT1080 cells expressing eIF2α WT and HA-eIF2α KIL-glutamine in the presence of 2 μg ml−1 puromycin (Sigma-Aldrich). TSC WT and knockout MEFs were maintained in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 1% L-glutamine. All culture media and reagents were obtained from Wisent Inc, unless specified. All cell lines were maintained in 5% CO2 at 37 °C. Micoplasma contamination was excluded using Mycoplasma PCR Detection Kit . Where indicated, cells were treated with 250 nM torin1 (Tocris), 50 nM rapamycin , 3 μM Ku-0063794 (Tocris), 10–50 μM CX-4945 (Adooq Bioscience), 1 μM thapsigargin (Sigma-Aldrich) or 1 μM salubrinal (Tocris). All drugs were stored in aliquots at −80 °C in DMSO for no longer than 6 months, and equal volume of DMSO was used as a vehicle control. HEK293E cells were freshly obtained from ATCC before the experiments (293 c18 (ATCC CRL-10852)), whereas the rest of the cell lines used were verified by profiling 17 short tandem repeat (STR) and 1 gender determining loci using ATCC Cell Line Authentication Service and the results were as following:HEK293E and MCF7 cells were obtained from American Type Culture Collection (ATCC) and maintained in DMEM and RPMI-1640, respectively, supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 1% HCT116 (94% match to ATCC HCT116 # CCL-247): D5S818 ; D13S317 ; D7S820 ; D16S539 ; vWA ; THO1 ; AMEL (X versus XY); TPOX , CSF1PO .MCF7 (100% match to ATCC MCF7 cells # HTB-22): D5S818 ; D13S317 (11 versus 11); D7S820 ; D16S539 ; vWA ; THO1 (6 versus 6); AMEL (X versus X); TPOX , CSF1PO (10 versus 10).U2OS (93% match to ATCC U2OS cells # HTB-96): D5S818 ; D13S317 (13 versus 13); D7S820 ; D16S539 ; vWA ; THO1 ; AMEL (X versus X); TPOX , CSF1PO .HT1080 (93% match to ATCC HT1080 cells # CCL-121): D5S818 ; D13S317 ; D7S820 ; D16S539 ; vWA ; THO1 (6 versus 6); AMEL ; TPOX (8 versus 8), CSF1PO (12 versus 12).TSC2 WT and knockout MEFs were generated by Kwiatkowski's group and obtained from Sonenberg's lab.E. coli transformed by heat shock with ∼300 ng of pGEX-6p-1-eIF2β constructs A, TOS and SD eIF2β mutants) was induced with isopropylthiogalactoside for 3 h and then lysed in lysis buffer (50 mM Tris HCl (pH 7.5), 300 mM NaCl, 10 μM TCEP (tris(2-carboxyethyl)phosphine; Sigma-Aldrich)). Lysates were incubated with 20 μl of Glutathione Sepharose 4B  for 1 h at 4 °C and then washed twice with lysis buffer supplemented with 5% glycerol and 0.1% NP-40, and 700 mM NaCl. Recombinant proteins were then washed once in buffer B . For in vitro translation and circular dichroism experiments, glutathione S-transferase (GST) tag was removed with PreScission protease . GST tag was not removed for the in vitro kinase assay.Human eIF2β was subcloned from pOTB7 vector (provided by Dr Kimchi) in pEF-FLAG vector (generously provided by Dr Ronai) using SalI and BamHI enzymes. Ser2 and Ser67 were mutated into an alanine (A) or aspartate (D) and phenylalanine 89 into an alanine (TOS mutant) using Quick-Change Site-Directed Mutagenesis (Agilent Technologies). For stable expression in mammalian cells, FLAG-eIF2β variants were subcloned in pWPI-GFP vector (Addgene) using PmeI enzyme. To produce the recombinant proteins, eIF2β was subcloned in pGEX-6p-1 vector  using BamHI and EcoRI. Recombinant proteins were purified as follows: 50 ml of BL21 (DE3) competent 6 HEK293T cells with 7 μg of each lentivirus packaging plasmids PLP1, PLP2 and PLP-VSVG (Invitrogen). Viral supernatant was collected 48 h post transfection, filtered through a Mixed Cellulose Ester filter , mixed in 1:1 ratio with growth media and added to cells for 24 h. Infection was carried out in the presence of 8 μg ml−1 of polybrene (Sigma-Aldrich). Forty-eight hours post infection, cells were selected and maintained in full growth media supplemented with 5 μg ml−1 puromycin. To generate cells lines expressing FLAG-eIF2β variants, viruses were generated and HEK293E cells were infected as described above. Expression of shRNA-insensitive eIF2β variants was monitored 72 h post infection by western blotting and cells were then infected with viruses carrying shRNA targeting eIF2β or scrambled control. Forty-eight hours post infection, cells were selected with 2 μg ml−1 puromycin as described above and expression of indicated exogenous eIF2β variants as well as depletion of endogenous eIF2β was determined by western blotting (Scrambled control (NS1) and siRNA targeting human NCK1 (5′-AACAUCCAUUACAUCUCCUUUCUCGAA-3′) were obtained from Integrated DNA Technologies. siRNA were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions at the final concentration of 10 nM. Cells were lysed 72 h post transfection in RIPA buffer and analysed by western blotting. Lentiviral vectors carrying shRNA targeting human raptor (plasmid 1857), human rictor (plasmid 1854) and the non-target shRNA control (plasmid 1864) were from Addgene. shRNAs targeting human eIF2β (TRCN0000291996) and scrambled control (SHC002) were from Sigma-Aldrich (Mission collection). Vectors encoding shRNAs (7 μg) were co-transfected into 5 × 10blotting .For western blotting, cells were scraped in 1 × PBS (pH 7.4), centrifuged and lysed in RIPA buffer (10 mM Tris (pH 7.3), 1% (w/v) Na-deoxycholate, 1% (v/v) Triton, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 10 mM β-glycerophosphate and protease inhibitors) supplemented with complete protease inhibitors (Roche). Whole-cell protein extracts (10–60 μg) were analysed by SDS–polyacrylamide gel electrophoresis  and transferred to nitrocellulose membrane (Bio-Rad) using wet transfer apparatus (Cleaver). Following antibodies were diluted as indicated in 5% (w/v) bovine serum albumin (Sigma-Aldrich) in 1 × TBS-Tween 20 and incubated overnight at 4 °C: anti-eIF2α (L57A5) #2103 , anti-p-eIF2α (S51) #9721 , anti-4E-BP1(53H11) #9644 (1:2000), anti-p-4E-BP1/2 (S37/46)(236B4) #2855 , anti-p-4E-BP1S65)(174A9) #9456 , anti-p70 S6 kinase #9202 , anti-p-p70 S6K1/2 (T389/388) #9205 , anti-p-rpS6 (S240/244) #2215 , anti-p-AKT (S473) #9271 , anti-AKT (pan) (C67E7) #4691 , anti-NCK1 (15B9) #2319 , anti-raptor (24C12) #2280 dilution , anti-rictor #2140 ; anti-PTEN (138G6) #9559 , anti-mTOR (7C10) #2983 , anti-PRAS40 #2610 , anti-p-PTEN  (44A7) #9549 , anti-PERK # 5683 , anti-p-PKR (T446) # 3076 , anti-eIF4G1 #2858  and anti-Myc-Tag #2276  all from Cell Signaling Technologies; anti-TSC2 sc-893 (1:500), anti-eIF2β (P-3) #sc-9978 , anti-ATF4 (CREB-2) C-20 #sc-200 , rpS6 (C-8) #sc-74459  and anti-p-PERK (T981) sc-32577 (1:500) all from Santa Cruz Biotechnologies; anti-eEF1δ #A301–685A  and eIF2Bδ #A302–982A  both from Bethyl Labs; anti-FLAG(M2) #F3165  and anti-β-actin (AC15) #A1978  both from Sigma; anti-eIF4E #610269  from BD Biosciences; anti- #ab9110  from Abcam; anti-PKR (F9)(174A9) #in vitro kinase assay, HEK293E cells were transfected in a 10-cm Petri dish with 7 μg of HA-Raptor using Lipofectamine 2000 (Invitrogen) according to manufacturer's instruction. HA-immunoprecipitation was performed in CHAPS buffer (40 mM HEPES KOH (pH 7.4), 2 mM EDTA, 10 mM sodium pyrophosphate, 0.1 M NaCl, 0.3% CHAPS), washed twice with CHAPS buffer and once in CHAPS buffer supplemented with 0.4 M NaCl. Beads were then washed twice in kinase buffer (25 mM HEPES KOH (pH 7.5), 50 mM KCl, 10 mM MgCl2) and resuspended in 40 μl of kinase buffer. One-sixth of the immunoporecipitated material and 1 μg of recombinant GST-eIF2β variants were used per reaction. Reaction was carried out at 30 °C for 30 min in a final volume of 50 μl, in the presence of 100 μM ATP, 0.5 μl of 10 μCi ml−1 of 32P-γATP (Perkin Elmer) and 1 × of kinase buffer. Reactions were stopped with 4 × Laemmli Sample Buffer and samples were loaded on a 10% SDS–PAGE gel. SDS–PAGE gel was then stained with Coomassie brilliant blue R-250, dried for 2 h at 80 °C and the phosphorylation was measured by 32P incorporation using Storm 860 Molecular Imager. Each experiment was carried out at least in two independent replicates.For In vitro translation assay and in vitro transcription of the reporter renilla mRNA were performed by depleting eIF2 using anti-eIF2β antibody. eIF2β was first linked to 10 μl of protein G agarose beads in 1 × at 4 °C for 2 h. Beads and antibody were then washed twice in PBS and twice in buffer D (25 mM HEPES KOH (pH 7.3), 50 mM KCl, 75 mM KOAc, 2 mM MgCl2). After the last wash, buffer D was removed and the antibody-conjugated beads were incubated with nuclease-treated RRL for 2 h at 4 °C. Purified eIF2α and eIF2γ lacking the eIF2β subunit were kindly provided by Yuri Svitkin from Sonenberg's laboratory. For translation reaction, 50 ng of the reporter mRNA was added to 7 μl of RLL in a final volume of 10 μl. Titration experiments were performed adding increasing amount of eIF2 β recombinant proteins . In eIF2-depleted RRL, eIF2 was reconstituted by adding 37.5 or 75 ng of purified eIF2α/γ subunits and 37.5 or 75 ng of recombinant eIF2β. Translation reaction was carried out at 30 °C for 30 min and stopped adding equal volume of 2 × PBS. Renilla expression was measured in 3 μl of RRL with 50 μl of Renilla Luciferase Assay system (Promega).5 cells were seeded in a six-well plate in triplicate and the luciferase assay was performed at 48 h post transfection with cells at 80% confluency. Cells were collected in Passive Lysis Buffer, and firefly and renilla luciferase activity was measured using the Dual-luciferase Reporter Assay kit (Promega) according to the manufacturer's instructions. Experiments were repeated three times independently (n=3), whereby each biological replicate consisted of a technical duplicate.Translation of the reporter mRNAs harbouring 5′UTR of human ATF4 ref. . was mon2, 1.5 mM KCl, 100 μg ml−1 cycloheximide, 2 mM dithiothreitol (DTT), 0.5% Triton, 0.5% sodium deoxycholate). Ten per cent of the lysates was saved to isolate cytoplasmic mRNA. The amount of RNA in each lysate was measured at 254 nm and 12 ODs were loaded on 5–50% sucrose gradients generated using Gradient Master (Biocomp) and subjected to ultracentrifugation . Sucrose gradients were fractionated by displacement by 60% sucrose/0.01% bromphenol blue, using ISCO Foxy fraction collector (35 s for each fraction=750 μl per fraction) equipped with a ultraviolet lamp for continuous absorbance monitoring. Fractions were flash-frozen immediately after fractionation and stored at −80 °C. RNA was isolated with Trizol (Thermo Fisher Scientific) according to the manufacturer's instruction. For microarray analysis, fractions corresponding to heavy polysomes (more than three ribsomes) were pooled (www.r-project.org) and bioconductor version 3.0. We used updated probe set definitions2 fold changes between insulin+torin1 versus insulin and insulin versus control for each RNA separately (polysome associated or cytosolic). Two-sided Wilcoxon rank-sum test was used to compare differences in fold changes between mRNAs whose translation was stimulated by phosphorylation of eIF2αhttp://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_040980.pdf. Experiments were performed at least in independent duplicates (n=2), whereby every sample was analysed in a technical triplicate. Primers were designed using NCBI Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) such that Tm was between 57 and 63 °C, Tm difference was <3 °C and that primer pairs were separated by at least one intron. Primers were obtained from Integrated DNA Technologies, and their sequences and size of the amplicons are listed below:Polysome profilinge pooled . RNA wasATF4 ; Amplicon=226 ntHuman ATF4-Forward 5′-TCAAACCTCATGGGTTCTCC-3′Human ATF4-Reverse 5′-GTGTCATCCAACGTGGTCAG-3′Homo sapiens ATF4, transcript variant 1, mRNA (NM_001675.4) and Homo sapiens ATF4, transcript variant 2, mRNA (NM_182810.2), both of which contain inhibitory uORFs and are translationally activated when eIF2α phosphorylation is induced and encode identical protein68Note: these primers recognize β-Actin ; Amplicon=163 ntActin HF 5′- ACCACACCTTCTACAATGAGC-3′Actin HR 5′- GATAGCACAGCCTGGATAGC-3′35S-methionine/cysteine labelling, HEK293E cells were seeded in six-well plates, serum starved for 16 h and then deprived of methionine and cysteine for an additional 2 h using DMEM without the above amino acids (Gibco). Cells were then treated with 10% dialysed serum containing 10 μCi ml−1 of 35S-Met/Cys (Perkin Elmer) and 250 nM of torin1 were indicated. Cells were lysed in RIPA buffer without SDS and 10 μl of the protein extract measured by LS6500 Multi Purpose Scintillation Counter (Beckman Coulter). Experiments were performed in independent triplicates (n=3) each of which was performed in a technical duplicate.For g for 10 min at 4 °C). m7GTP-Agarose beads  were equilibrated in buffer C (50 mM MOPS KOH (pH 7.4), 100 mM NaCl, 50 mM NaF, 0.5 mM EDTA, 0.5 mM EGTA, 7 mM β-mercaptoethanol, 2 mM benzamidine or 0.5 mM PMSF, 1 mM Na3VO4 and 0.1 mM GTP (Sigma-Aldrich)). After equilibration, lysates were diluted  to 1 ml with buffer C (in a 2-ml tube) and incubated with equilibrated m7GTP-Agarose beads (∼50 μl of 50% slurry) for 20 min at 4 °C end-over-end rotation. Ten per cent of the lysate was used as the input. The beads were collected by centrifugation (500g for 5 min at 4 °C) and washed four times with 1.5 ml of buffer C. Bound proteins were eluted with 0.2 mM m7GTP, resuspended in SDS–PAGE loading buffer and analysed by western blotting along with the inputs. Experiments were performed in independent duplicate.MCF7 and HCT116 cells were treated as described in −1 reversible crosslinker 3,3′-dithiobis (sulfosuccinimidylpropionate) DTTSP (Life Technologies). Two microlitres of anti-raptor antibody  and 30 μl of protein G-sepharose 50% slurry (Millipore) were equilibrated in CHAPS buffer and incubated in 100 μl of CHAPS buffer for 30 min at 4 °C, washed followed by incubation with 1 mg of the lysates for 4 h at 4 °C, with end-to-end rotation. Beads were washed four times with 1 ml of ice-cold CHAPS buffer and collected by centrifugation (500g for 2 min at 4 °C). Fifty microlitres of beads were resuspended in the sample buffer, boiled and analysed by western blotting.Raptor immunopreciptations were carried out using an anti-raptor antibody (Millipore). HEK293E cells were serum starved overnight and then treated as described in figure legends. Cells were collected in CHAPS buffer , 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM β-glycerophosphate, 50 mM NaF, 1 mM DTT). Immunoprecipitation was performed in the presence of 0.5 mg mlg at 4 °C. An amount of 1 mg ml−1 of the protein was incubated with 40 μl of the NET-2 buffer equilibrated protein A sepharose beads  on which the supernatant was split in two, set at 500 μg ml−1 and incubated for 2 h at 4 ° C (end-over-end rotation) with either eIF2β antibody (P-3) #sc-9978 from Santa Cruz Biotechnologies or the appropriate IgG1 control . On the incubation with the antibody, protein A sepharose beads were added (∼30 μl μg−1 of antibody), and the incubation was carried out for the additional 2 h at 4 °C. After the immunoprecipitated beads were washed one time with NET-2 buffer containing 300 mM NaCl and five times with NET-2 buffer containing 150 mM NaCl. Beads were eluted in Laemmli buffer by boiling and eluates were analysed by western blotting.For endogenous eIF2β immunoprecipitations, cells were collected by scraping, washed three times in ice-cold PBS  and lysed in NET-2 buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 2 mM MgOAc, 0.1% NP-40, 1 mM DTT, 1 × EDTA-free protease inhibitors (Roche)) by 3 × 10-s bursts using microtip at power 6 on ice. The lysates were spun for 10 min/16,1002, 0.1 mM EDTA, 5 mM EGTA, 1 mM DTT, 20 mM NaF, 0.1 mM Na3VO4, 20 mM β-glycerophosphate, 0.3% CHAPS). FLAG-eIF2β variants were immunoprecipitated using 10 μl FLAG-agarose beads (Sigma-Aldrich), previously equilibrated in eIF2-IP buffer, for 90 min at 4 °C. Immunoprecipitated material was washed twice in 1 ml of eIF2-IP buffer . FLAG-eIF2β was eluted three times with FLAG-eluting peptide at 4 °C according to the manufacturer's instructions. To monitor eIF2:tRNA binding, half of the immunoprecipitated material was analysed by western blotting and half was used to extract RNA using Trizol. Analysis of tRNAiMet and tRNALys was performed by semi-quantitative reverse transcription–PCR (sqRT–PCR) using OneStep RT–PCR kit (Qiagen) and quantitative reverse transcription–PCR (qRT–PCR) using Power SYBR Green RNA-to-Ct 1-Step Kit (Applied Bioscience) according to the manufacturer's instruction. Following primers were used:Anti-FLAG immunopreciptiations were carried out using anti-FLAG(M2) #F3165 and the same concentration of isotype-matched mouse IgG1 antibody #M5284 as a control (both from Sigma). Two micrograms of the antibody/IgG per 100–500 μg of total cell protein were used. HEK293E cells were seeded in a 15-cm Petri dish and collected at 80% confluency in 1.5 ml eIF2-IP buffer (20 mM Tris HCl (pH 7.5), 100 mM KCl, 5 mM MgCliMet_; Amplicon: 71tRNAiMet_Fwd: 5′-AGCAGAGTGGCGCAGCGGAAGCGTGCT-3′tRNAiMet_Rev: 5′-TAGCAGAGGATGGTTTAGATCCATC-3′tRNALys_; Amplicon: 72tRNALys_Fwd: 5′-GCCCGGATAGCTCAGTCGGTAGA-3′tRNALys_Rev: 5′-CGCCCGAACAGGGACTTGAACC-3′tRNAT=60 °C for 30 s, denaturation T=94 ° C for 1 min and elongation T=72 °C for 1 min for 30 cycles. qRT–PCR reactions were carried out under the following conditions: reverse transcription: 48 °C for 30 min; activation of DNA polymerase: 95 °C for 10 min; 40 cycles: denature 95 °C for 15 s; anneal/extend 60 °C for 1 min. All immunoprecipitation/PCR experiments were carried out at least in independent duplicates.sqRT–PCR reactions were carried out using the following conditions: annealing 4) expressing eIF2β variants, in which endogenous eIF2β was depleted by shRNA (4 cells were seeded in a 96-well plate and maintained in DMEM without amino acids (Gibco) supplemented with 10% dialysed serum for 6 and 18 h (Invitrogen). Proliferation rate was determined using Cell Proliferation ELISA BrdU kit (Roche). Absorbance at 370 nm (reference wavelength 492 nm) was measured using a Benchmark Plus microplate reader (Bio-Rad). The experiments were performed in independent triplicate (n=3), with four technical replicates per biological replicate.HEK293E cells A, TOS and SD eIF2β mutants (4 μg in 100 μl of 10 mM phosphate buffer (pH 7.2), 50 mM NaCl) purified as described in the ‘Constructs and recombinant proteins' section were continuously detected from 197 to 250 nm using Chirascan CD Spectrometer (Applied Photophysics). The measurements were carried out in using a 1-mm path-length cuvette (Hellma) at room temperature using a 1-nm bandwidth. For each sample, two spectra were collected and averaged. The spectral contribution of the buffer was corrected for by subtraction.TOS motifs (F E/D/V M/I/L D/E/V I/E/L) were identified in RefSeq proteins using pattern matching2, 0.1% Na-deoxycholate, 100 μg ml−1 cycloheximide, 1 mM DTT, 20 mM NaF, 0.1 mM Na3VO4 and 20 mM β-glycerophosphate. Five ODs (at 254 nm) were loaded onto 15–35% sucrose gradients buffered with 20 mM HEPES (pH 7.6), 100 mM KCl, 5 mM MgCl2, 100 μg ml−1 cycloheximide, 1 × protease inhibitor cocktail (EDTA free), 100 U ml−1 RNase inhibitor (Ambion) and subjected to ultracentrifugation . Absorbance profiles were detected as described in the section entitled ‘Genome-wide polysome profiling and qRT–PCR'. Area under the monosome (80S) peak areas were integrated using ImageJ, and presented as 80S vector control versus indicated eIF2β variants.For ribosome profiles shown in Accession codes: Raw and processed data are available at the Gene Expression Omnibus .How to cite this article: Gandin, V. et al. mTORC1 and CK2 coordinate ternary and eIF4F complex assembly. Nat. Commun. 7:11127 doi: 10.1038/ncomms11127 (2016).Supplementary Figures 1-10


Four highly pure steroid derivatives (1–4) were isolated from the dichloromethane fraction and water layer of P. nodosus. Potent inhibitory activities were also observed for (25S) 5α-cholestane-3β,4β,6α,7α,8β,15α,16β,26-octol (3) on the production of IL-12 p40 and IL-6 (IC50s = 3.11 ± 0.08 and 1.35 ± 0.03 μM), and for (25S) 5α-cholestane-3β,6α,8β,15α,16β,26-hexol (1) and (25S) 5α-cholestane-3β,6α,7α,8β,15α,16β,26-heptol (2) on the production of IL-12 p40 (IC50s = 0.01 ± 0.00 and 1.02 ± 0.01 μM). Moreover, nodososide (4) exhibited moderate inhibitory effects on IL-12 p40 and IL-6 production.The methanolic extract and dichloromethane fraction exerted potent inhibitory effects on the production of all three pro-inflammatory cytokines, with ICP. nodosus. The main finding of this study is the identification oxygenated steroid derivatives from P. nodosus with potent anti-inflammatory activities that may be developed as therapeutic agents for inflammatory diseases.This is the first report of the anti-inflammatory activity from the starfish  Inflammation is one of the earliest innate immune responses to tissue injury and various pathological stimuli. It is also a critical early factor in wound healing. Delayed or impaired inflammation may affect the later stages of wound healing, especially granulation formation ,2. InflaInterleukin (IL)-12 is a pro-inflammatory cytokine produced by activated antigen-presenting cells, dendritic cells, monocytes/macrophages, and B cells in response to bacterial products and immune signals . OriginaTNF has since been implicated in diverse inflammatory, infectious, and malignant conditions, and the importance of TNF in inflammation was demonstrated by the efficacy of anti-TNF antibodies or the administration of soluble TNF receptors (TNFRs) in controlling rheumatoid arthritis and other inflammatory conditions ,11. TNF Starfish are found in all oceans. There are more than 1,500 known species, and many remain undiscovered. Forcipulatida, Paxillosida, Platyasterida, Spinulosida, and Valvatida are the main subclasses of Asteroidea. Starfish have been investigated by organic chemists, biochemists, and pharmacologists as a potential source of bioactive marine natural products. Various secondary metabolites, including steroids, steroidal glycosides, anthraquinones, alkaloids, phospholipids, peptides, and fatty acids, have been reported in starfish . SteroidProtoreaster nodosus  is an invertebrate belong to the order Phanerozonia, class Asteroidea, and phylum Echinodermata. Members of the genus Protoreaster are found in the warm Vietnamese sea, and have been historically used as tonic agents in Vietnamese folk medicine. However, studies on the biological activities of P. nodosus are limited. In previous studies, the main constituents of P. nodosus were found to be polyhydroxylated steroids [The starfish steroids , severalsteroids , galactosteroids , and gansteroids .P. nodosus showed significant in vitro anti-inflammatory effects. The present study details the inhibitory effect of steroid derivatives  and processed foods. Oxysterols are emerged as intriguing substances with diverse biological activities [Natural marine products have recently become the focus of increased research interest due to their potential pharmacological activities and low-level toxicity ,33. Oxystivities ,35. Recetivities , which ptivities ,36.BMDCs play a key role in the interface between the innate and acquired immune systems. Activated BMDCs perform crucial functions in immune and inflammatory responses via the pathogen-associated molecular patterns (PAMPs)-stimulated production of pro-inflammatory cytokines such as IL-12 p40, IL-6, and TNF-α, which are involved in the pathogenesis of cardiovascular and neurodegenerational diseases and cancers through a series of cytokine signaling pathways. It has been demonstrated that pro-inflammation is associated with pathophysiology and is connected with various clinical disease manifestations. These pro-inflammatory cytokines play a crucial role in host defenses and inflammation ,38.Among its many biological activities, IL-12 provides an obligatory signal for the differentiation of effector T-helper type 1 (Th1) cells and the secretion of Th1 cytokines, gamma interferon (IFN-γ), and IL-2. IL-12 plays an important role in the generation of Th1 responses to human pathogens ,24. AlthP. nodosus have not been reported. Therefore, we assayed the anti-inflammatory activity of a methanolic extract, dichloromethane-soluble fraction, and water layer on pro-inflammatory cytokine  production in LPS-stimulated BMDCs using ELISA . Since the methanolic extract significantly reduced inflammation, it was partitioned in dichloromethane/water to obtain a dichloromethane-soluble portion and an aqueous phase. As shown in Table 50s = 0.60 ± 0.01 and 3.29 ± 0.09 μg/mL, respectively), which was greater than in the presence of the methanolic extract (IC50s = 2.48 ± 0.08 and 8.57 ± 0.21 μg/mL). Moreover, the aqueous layer exerted potent suppressive effects on the production of IL-12 p40 (IC50 = 4.03 ± 0.10 μg/mL).The methanolic extract significantly inhibited pro-inflammatory cytokine production, while the dichloromethane fraction and water layer showed more potent inhibitory effects Figure . The met(1–4) from the dichloromethane fraction and water layer of P. nodosus were tested for inhibitory effects on the production of the pro-inflammatory cytokines IL-12 p40, IL-6, and TNF-α. The results obtained with 3--2,5 diphenyl tetrazolium bromide (MTT) assays showed that steroid derivatives (1–4) did not exhibit significant cytotoxicity at concentrations up to 50.0 μM at 24 h (data not shown). Of the tested compounds, 1–3 exerted greater inhibitory effects than SB203580 on IL-12 p40 production with IC50 values of 0.01 ± 0.00, 1.02 ± 0.01, and 3.11 ± 0.08 μM, respectively, at the various concentration  5α-cholestane-3β,6α,8β,15α,16β,26-hexol (1) is similar to that of (25S) 5α-cholestane-3β,6α,7α,8β,15α,16β,26-heptol (2) and (25S) 5α-cholestane-3β,4β,6α,7α,8β,15α,16β,26-octol (3), except for the presence of hydroxy groups located at C-4 and/or C-7 in compounds 2 and 3; these compounds had the greatest inhibitory activity towards LPS-stimulated IL-12 p40 production (IC50 = 0.01 ± 0.00 μM), which was comparable to that of the positive control, SB203580 (IC50 = 5.00 ± 0.16 μM). Previously, compounds 1–3 were isolated from Asterina pectinifera, Pentaceraster alveolatus, and Oreaster reticulatus [in vitro [(4) is a polyhydroxylated steroid glycoside. Structurally, these glycosides are oxygenated derivatives of cholesterol that are glycosylated with a disaccharide chain, but 4 exhibited a moderate effect (IC50 = 12.47 ± 0.38 μM) up to 50.0 μM, relative to the vehicle group. Our finding that steroid derivatives (1–3) possess significant anti-inflammatory activity suggests that these related steroids might be interesting for further investigations for potential inflammatory diseases.Comparing the observed effects between the structurally steroid derivatives, . (25S) 5α-cholestane-3β,4β,6α,7α,8β,15α,16β,26-octol (3) inhibited IL-6 production significantly in LPS-stimulated BMDCs, with an IC50 value of 1.35 ± 0.03 μM (Table (4) included moderate inhibition of IL-6 production, with an IC50 value of 23.18 ± 0.46 μM  against IL-6 production.IL-6 is a pro-inflammatory cytokine that induces inflammatory upon secretion by T cells and macrophages in response to trauma, especially burns or other types of tissue damage . It also50s = 26.19 ± 0.64 and 10.29 ± 0.34 μg/mL, respectively). Notably, not all of the isolated steroid derivatives (1–4) had significant inhibitory effects on TNF-α production , suggesting the presence of other active derivatives, which could act individually or by synergy with the isolated steroid derivatives (1–4).Overexpression of the pro-inflammatory cytokines TNF-α and IL-6 is associated with the development of autoimmune, inflammatory, and immunopathological diseases. Therefore, blocking TNF-α, IL-6, and their respective signaling pathways may be effective for the treatment of inflammatory diseases. According to our results, the methanolic extract and dichloromethane-soluble fraction showed inhibitory effects on LPS-stimulated TNF-α production  5α-cholestane-3β,4β,6α,7α,8β,15α,16β,26-octol (3) on the production of IL-12 p40 and IL-6, and for (25S) 5α-cholestane-3β,6α,8β,15α,16β,26-hexol (1) and (25S) 5α-cholestane-3β,6α,7α,8β,15α,16β,26-heptol (2) on the production of IL-12 p40. Moreover, nodososide (4) exerted moderate inhibitory effects on IL-12 p40 and IL-6 production. Additional studies are required to assess the mechanisms of action of these compounds and their potential use as novel anti-inflammatory agents. These results support the use of starfish steroid components to inhibit the secretion of pro-inflammatory cytokines, including IL-12 p40, IL-6, and TNF-α, and to prevent and treat inflammatory diseases.In conclusion, the methanolic extract and dichloromethane fraction showed potent inhibitory effects on the production of all three pro-inflammatory cytokines, with IC1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were recorded on a Bruker AM500 FT-NMR spectrometer and TMS was used as an internal standard. Column chromatography (CC) was performed on silica gel , porous polymer gel , octadecyl silica , and YMC RP-18 resins . Thin layer chromatography (TLC) used pre-coated silica gel 60 F254  and RP-18 F254S plates  and compounds were visualized by spraying with aqueous 10% H2SO4 and heating for 3–5 minutes.Optical rotations were determined on a JASCO P-2000 polarimeter. The UV spectrum was recorded on a JASCO V-630 spectrophotometer. IR spectra were obtained on a Bruker TENSOR 37 FT-IR spectrometer. The P. nodosus was collected at Ha Long, Quangninh, Vietnam, in October 2012 and identified by Prof. Do Cong Thung . A voucher specimen (PN-VHS-2012) was deposited at the Institute of Marine Resources and Environment and Institute of Marine Biochemistry, VAST, Vietnam.The sample of the starfish P. nodosus (5.0 kg) were ground and extracted three times with hot MeOH (at 50°C for 3 h each time). The obtained solutions were filtered, combined, and concentrated under reduced pressure to yield a dark viscous residue . This residue was suspended in water (1.0 L) and partitioned in turn with CH2Cl2 (3 × 1.0 L). The combined dichloromethane-soluble portions were evaporated under reduced pressure to afford CH2Cl2 fraction  and water layer (C).Fresh frozen samples of the starfish n-hexane from 0 to 100% to yield four fractions (B-1 to B-4). Fraction B-4 (2.31 g) was further separated by silica gel CC using CH2Cl2-MeOH-H2O (5:1:0.1) as eluents, to give three subfractions (B-1.1 to B-1.4). Subfraction B-1.3 (1.35 g) was then subjected to silica gel CC using eluent of CH2Cl2-MeOH (3.5:1), and further purified by reversed-phase flash CC  and eluting with MeOH-H2O (2:1) to afford (25S) 5α-cholestane-3β,6α,8β,15α,16β,26-hexol . Next, (25S) 5α-Cholestane-3β,6α,7α,8β,15α,16β,26-heptol  was purified via the subfraction of B-1.2 (0.21 g) by silica gel CC and eluting with CH2Cl2-MeOH (4.5:1). Similarly, subfraction B-1.1 (4.16 g) was subjected to YMC RP-18 CC, using MeOH-acetone-H2O (80:10:10) to yield (25S) 5α-cholestane-3β,4β,6α,7α,8β,15α,16β,26-octol .Extract B was crudely separated by silica gel (CC) using gradient concentrations of ethyl acetate (EtOAc) in 2Cl2-MeOH  to yield five fractions (C-1 to C-5). Fraction C-3 (1.2 g) was separated by silica gel CC, using CH2Cl2-MeOH (5:1) as the eluent to yield three subfractions (C-3.1 to C-3.3). Finally, subfraction C-3.3 (0.15 g) was further separated by Sephadex® LH-20 CC using acetone-MeOH (1:2) as the mobile phase, followed by YMC RP-18 CC using MeOH-H2O (1:1) to afford nodososide .The water layer was desalted by Diaion™ HP-20 CC and eluted first with water and then with MeOH. The desalted water residue  was separated by silica gel CC and eluted with CHin vacuo at ambient temperature for 24 h. From the dichloromethane fraction and water layer of P. nodosus, four highly pure steroid derivatives (1–4) were isolated and their structures elucidated. Stock solutions of the tested compounds were prepared in dimethyl sulfoxide (DMSO), kept at −20°C, and diluted to the desired final concentration in fresh medium before each experiment. The final concentration of DMSO did not exceed 0.5% in any of the experiments to avoid effects cell growth.In total, 5.0 kg of finely chopped material were extracted with MeOH (5.0 L) in glass jars with screw-on lids. The methanolic extracts were filtered, combined, and concentrated using a rotary vacuum evaporator at 40°C and then further dried 4 cells were seeded on a 96-well culture plate. After incubation for 1 h at 37°C, cells were treated with extract, fractions, and compounds at various concentrations for 24 h. Cells were added 0.2 mg MTT  and then incubated at 37°C for 4 h. The plate was centrifuged and the supernatants were aspirated. The formazan crystals in each well were dissolved in 250 μL DMSO . The plates were agitated to ensure complete dissolution of the purple formazan crystals, and the optical density was measured at the wavelength 540 nm using an ELISA reader .The cell viability was determined by standard procedure of MTT assays , which was based on the reduction of the dye MTT to formazan crystals, an insoluble intracellular blue product, by cellular dehydrogenases. Briefly, the cells at a concentration of 5 × 105) were seeded in 48-well plates at 37°C, 5% CO2 for 1 h, and then treated for 1 h with the compounds at concentrations of 0.1 to 50.0 μM, followed by stimulation with LPS  as previously described . All ani5 cells per well, and then treated with the isolated compounds (1–4) at different concentrations for 1 h before stimulation with 10.0 ng/mL LPS from Salmonella minnesota . Supernatants were harvested 18 h after stimulation. Concentrations of murine IL-12 p40, IL-6, and TNF-α, in the culture supernatant were determined by ELISA  according to the manufacturer’s instructions. The data are presented as means ± S.D. of at least three independent experiments performed in triplicate.The BMDCs were incubated in 48-well plates in 0.5 mL containing 1 × 10IL-12 p40 level in unstimulated DC: not detectable. IL-12 p40 level in LPS-stimulated DC: 51.34 ± 0.66 (ng/mL). IL-6 level in unstimulated DC: not detectable. IL-6 level in LPS-stimulated DC: 41.12 ± 2.38 (ng/mL). TNF-α level in unstimulated DC: not detectable. TNF-α level in LPS-stimulated DC: 1.83 ± 0.02 (ng/mL).The inhibitory activity (I) was expressed as the inhibition rate (%), which was calculated from the following formula:Cdcv: Cytokine level (ng/mL) in vehicle treated DC; Cdcc: Cytokine level (ng/mL) in compound treated DC.P < 0.05 was considered statistically significant.The results are expressed as mean value ± S.D. Statistical analysis was performed using one-way ANOVA.


The title compound is a resolved chiral ester derivative of mandelic acid containing an acetate group and a carboxylic acid group, which engage in intermolecular hydrogen bonding, forming chains extending parallel to [001]. R)-(−)-2-acetoxy-2-phenylacetic acid], C10H10O4, is a resolved chiral ester derivative of mandelic acid. The compound contains an acetate group and a carboxylic acid group, which engage in intermolecular hydrogen bonding, forming chains extending parallel to [001] with a short donor–acceptor hydrogen-bonding distance of 2.676 (2) Å.The title compound [systematic name: ( R)-(−)-2-acetoxy-2-phenylacetic acid (I)R)-O-acetylmandelic acid, is a chiral, resolved derivative of mandelic acid. The compound may be synthesized by acetylation of the parent α-hydroxy acid with acetic anhydride in pyridine  and Hooft y = −0.02 (4), calculated with PLATON  −x + y + 1, z − c axis of the unit cell -[(2S)-2-hydroxy-2-phenylethanoyloxy]phenylacetic acid -2-[(R)-mandeloxycarbonyl]bicyclo(2.2.1)heptane-3-carboxylic acid -2-[2-(3-methoxy-3-oxoprop-1-en-1-yl)-4-(trifluoromethyl)phenyl]-2-acetic acid -(−)-2-acetoxy-2-phenylacetic acid (99%) was purchased from Aldrich Chemical Company, USA, and was used as received. : δ 2.19 , 5.93 , 7.36–7.42 , 7.45–7.51 , 11.76 . 13C NMR : δ 20.59 (CH3), 74.02 (CH), 127.62 (CarylH), 128.86 (CarylH), 129.49 (CarylH), 132.98 (Caryl), 170.38 (CO), 174.55 (CO). IR : 3483 (w), 3014 , 2708 (w), 2588 (w), 1752 , 1686 , 1498 (w), 1461 (w), 1412 (m), 1382 (s), 1321 (m), 1277 (s), 1259 (s), 1206 (s), 1182 (s), 1045 (s), 996 (m), 967 (m), 919 (m), 888 (m), 767 (s), 734 (s), 700 (s), 642 (m), 616 (w), 603 (w), 583 (w), 525 (s), 487 (w). GC/MS (Hewlett-Packard MS 5975/GC 7890): M-18+ = 176 .Uiso(H) = 1.2, 1.5 and 1.2 × Ueq(C) of the aryl, methyl and methine C atoms, respectively. The position of the carboxylic acid hydrogen atom was found in the difference map and the atom refined semi-freely using a distance restraint d(O—H) = 0.84 Å, and Uiso(H) = 1.2Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016008653/pk2580sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989016008653/pk2580Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016008653/pk2580Isup3.cmlSupporting information file. DOI: 1482445CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


DDIAS, we determined the transcription start site, promoter region, and transcription factor. We found that DDIAS transcription begins at nucleotide 212 upstream of the DDIAS translation start site. We cloned the DDIAS promoter region and identified NFAT2 as a major transcription factor , or human Noxin (hNoxin), is strongly expressed in lung cancers. DDIAS knockdown induced apoptosis in non-small cell lung carcinoma A549 cells in response to DNA damage, indicating DDIAS as a potential therapeutic target in lung cancer. To understand the transcriptional regulation of  Specifications Table•DDIAS transcription.These data provide information regarding the transcription site and promoter used to control •DDIAS transcription in lung cancer cells.NFATc1 plays a crucial role in •NFATc1 also controls DDIAS expression in the pancreatic cancer cell line PANC-1, as demonstrated by growth inhibition in a knockdown model.1DDIAS translation start site or 32 base pairs (bp) downstream of the site reported in the National Center for Biotechnology Information (NCBI) database , a calcineurin inhibitor, phorbol 12-myristate 13-acetate (PMA), and the calcium ionophore A23187 (DDIAS mRNA (DDIAS mRNA level decreased after treatment with CsA (A 5′-rapid amplification of cDNA ends (RACE) assay revealed a transcription start site located 212 nucleotides upstream of the database . This trdatabase . Putativpromoter A. In a Ppromoter B and C. e A23187 . TreatmeIAS mRNA A. In conwith CsA B. Previowith CsA .22.1DDIAS, 5′ RACE was performed using the SMART RACE cDNA Amplification Kit  according to the manufacturer׳s protocol. First-strand cDNA was synthesized using RNA from human embryonic kidney 293 (HEK293) cells. 5′-RACE PCR was performed using the Advantage 2 PCR kit (Clontech) and the following gene-specific primers: R1; 5′-TGTACCTGTGCAAACCAGTGGCAGT-3′ or R2; 5′-AAAGCCTGCAATACCTGGGTCTGGT-3′. The PCR products were separated by electrophoresis on 1.5% agarose gels. After purification using the QIAquick Gel Extraction Kit . PCR products were subcloned into the pGEM-T vector using the TA Cloning Kit  and sequenced. The first base pair after the adapter was identified as the transcription start site of the human DDIAS gene To identify the 5′ UTR of human 2.22. HEK293 and Panc-1 cells were cultured in Dulbecco׳s modified Eagle׳s medium (DMEM); NCI-H1703 cells were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS), 50 U/mL of penicillin, and 50 μg/mL of streptomycin . Cells were transfected with 40 nM siRNAs with Lipofectamine 2000  according to the manufacturer׳s instructions. siRNA sequences were as follows: siSP1, 5′-CUUUUUCACCAAUGCCAAU-3′, sicJun: 5′-CAAGAAGAUGCGCCGCAAC-3′, sicMyc: 5′-GUCAAGAGGCGAACACACA-3′, siNFATc1: 5′-GGACUCCAAGGUCAUUUUC-3′, siDDIAS D1: 5′-GCCUAUCAUUUCCCUGAUC-3′, siDDIAS D2: 5′-CUGUAACCCAGGCAGAUGU-3′, or siScrambled: 5′-CCUACGCCACCAAUUUCGU-3′.HEK293, non-small cell lung cancer cell NCI-H1703, and pancreatic cancer cell Panc-1 cells were cultured in an incubator at 37 °C and 5% CO2.3DDIAS promoter (−1205) and pRL-TK, using 2 μl of Polyfect  per well. Cells were harvested 36 h after transfection and lysed in 1× passive lysis buffer. Firefly and Renilla luciferase activities were assayed using a dual-luciferase reporter assay system  and a luminometer . Firefly luciferase activity was normalized to Renilla luciferase activity and expressed as the relative luciferase activity (RLA) to reflect promoter activity.Cells were co-transfected with the 2.4Total RNA was extracted from cells using TRIzol (Invitrogen) and was reverse-transcribed into cDNA using TOPscript™ RT DryMIX (dT18)  according to the manufacturer׳s instructions. The cycling conditions were 95 °C for 5 min, 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The following primers were used: NFATc1 F, 5′-GGGTTAAGTCCTCTCCCAAG-3′; R, 5′-GGGTTAAGTCCTCTCCCAAG-3′; DDIAS F, 5′-CTTGCAGCAGTTGTTACGAA-3′; R, 5′-GTGACCAAGCACTTCGAGTT-3′; GAPDH F, 5′-TCATGACCACAGTCCATGCC-3′; R, 5′-TCCACCACCCTGTTGCTGTA-3′.2.5ChIP assays were performed as previously described 2.63VO4, 1 mM sodium fluoride, 1 mM PMSF, and protease inhibitor cocktail . The lysates were quantified using a BCA assay kit  and subjected to immunoblotting with specific antibodies, as follows: anti-SP1 (#5931) or anti-cJun (#9165) from Cell Signaling Technology , anti-cMyc (sc-40) or anti-NFATc1 (sc-7294 X) from Santa Cruz Biotechnology , anti-GAPDH (LF-PA0212) from AbFrontier . Antibodies were diluted in 5% BSA in phosphate-buffered saline containing 0.1% Tween 20 (PBS-T) overnight at 4 °C. After washing three times in PBS-T for 10 min, membranes were incubated for 1 h with horseradish peroxidase-labeled goat anti-mouse IgG (LF-SA8001) or goat anti-rabbit IgG (LF-SA8002) from AbFrontier and washed three times in PBS-T for 10 min. Western blot signals were detected using an enhanced chemiluminescence (ECL) kit (Millipore).Cells were lysed with 1X RIPA buffer (Millipore) containing 1 mM Na2.7Cell viability was determined using the sulforhodamine B assay, as previously described


Sophora flavescens by plate method, which has broad antimicrobial activity. Isolation and trace of a new bioactive compound from the fungus’ culture extracts with the method of column chromatography and TLC biological autoradiography was conducted. Finally, it was identified as 6,7-(2′E) dibutenyl-5,8-dihydroxy-(Z)-cyclooct-2-ene-1,4-dione by nuclear magnetic resonance, infrared and liquid chromatography–mass spectrometry. The compound presented strong antifungal activities for example: Botryosphaeria berengriana f.sp.Ell. Arthur., Fusarium oxysporum f.sp. cucumerinum, Fusarium moniliforme. piricola, Physalospora piricola, Cladosporium cucumerinum  The inhibition to Physalospora piricola was the strongest with an antibacterial diameter of 45 mm. This paper is the first report of the antimicrobial activity of endophytic fungi BS002 that was the secondary metabolites extracted from the seeds of Sophora flavescens. The results provide a broad foreground for biopharmaceuticals and biopesticide.Endophytic fungus BS002 was isolated and characterized from  Taxus mairei which had good antitumor effects.+ , given by HR-TOF-MS, and its molecular formula (C16H18O8) was determined. The infrared spectrum gave strong wide peak in νOH 3383 cm−1, a conjugate strong absorption peak νC=O in 1695 cm−1, and an absorption peak νC=C in 1633 cm−1. Ultraviolet spectrum λMax 309 nm (ϵ 3.20), and combined with its NMR, it could be speculated that there was a conjugated system containing unsaturated carbonyl in the structure of the molecule. The following was documented 1H-NMR : δ6.81 , δ6.43 , δ6.09 ; δ4.23 , δ4.82 ; δ1.90 ; δ6.81 , δ6.43 , δ1.90 . Also 13C-NMR  δ205.9 (C=O), δ170.9 (C=O); three carbon signals, δ143.5 (C=C), δ124.6 (C=C), δ124.5 (C=C); δ80.5 (C–O), δ76.1 (C–O); δ18.9 (–CH3). According to the combined data of NMR and mass spectrum it could be speculated that the compound is a symmetric structure.The compound with strong antimicrobial activity was identified as 6,7-(2′E)dibutenyl-5,8-dihydroxy-(Z)-cyclooct-2-ene-1,4-dione on the basis of the experimental results obtained in our laboratory. Its chemical structure was shown at 1-H1-COSY, DEPT, HSQC and HMBC (H 6.09) and C-4 (δC 205.9), C-5(δC 80.5); H-5 (δH 4.23) and C-3 (δC 124.6), C-6 (δC 76.1); H-6 (δH 4.82) and C-4 (δC 205.9), C-5 (δC 80.5), C-1′ (δC 170.9); H-2′ (δH 6.43) and C-1′ (δC 170.9); H-3′ (δH 6.81) and C-1′ (δC 170.9), C-2′ (δC 124.5); H-4′ (δH 1.90) and C-2′ (δC 124.5), C-3′ (δC 143.5). According to the wide singlet type of H-5 and H-6, it could be judged that H-5 and H-6 are in the same plane. This compound is the derivative of cyclooct-2-ene-ketone,[The structure of this compound was deduced by two-dimensional NMR technology, such as Hand HMBC . One of e-ketone, and nameS. flavescens by studing the secondary metabolites of the endophytic fungus BS002. However, the obtained substance with antimicrobial activity was different from the known compounds. It had a cyclic symmetric olefin structure with unsaturated double bonds and carbonyl group, and lively physical and chemical properties. At temperatures greater than 55 °C or under slightly alkaline conditions the compound easily lost its activity in the test. We believe that this compound with a broad-spectrum antifungal activity may be put to development as a new biopharmaceutical product.We expected to obtain some structures which were the same or similar with the active composition from Penicillium sp. M-01, a variant of Penicillium sp.) was isolated from the seeds of healthy S. flavescens for the first time, and the antibacterial substance was identified as 6,7-(2′E) dibutenyl-5,8-dihydroxy-(Z)-cyclooct-2-ene-1,4-dione. In this study, we obtained the compound from culture filtrates of BS002. The result provided a green process and a new way to produce this compound. Antimicrobial assay showed that endophytic fungus BS002 inhibited the growth of the tested 24 microorganisms, having a wider antimicrobial spectrum than the positive references. These results suggest that endophytic fungus BS002 has a potent antimicrobial activity and could be a valuable candidate for the discovery of new antimicrobial drugs.In this research, endophytic fungus BS002 (