However, high kinetic barriers lead to slow absorption/desorption rates even at relatively high temperatures (>180 °C). In this work, we investigate the effects of the addition of K-modified LixTiyOz on the absorption/desorption behaviour of the Mg(NH2)2 + 2LiH system. In comparison with the pristine Mg(NH2)2 + 2LiH, the system containing a tiny amount of nanostructured K-modified LixTiyOz shows enhanced absorption/desorption behaviour. The doped material presents a sensibly reduced (∼30 °C) desorption onset temperature, notably shorter hydrogen absorption/desorption times and reversible hydrogen capacity of about 3 wt.% H2 upon cycling. Studies on the absorption/desorption processes and micro/nanostructural characterizations of the Mg(NH2)2 + 2LiH + K-modified LixTiyOz system hint to the fact that the presence of in situ formed nanostructure K2TiO3 is the main responsible for the observed improved kinetic behaviour.The system Mg(NH However, storing 5 kg of hydrogen in a high-pressure (700 bar) tank requires an internal volume of 122 litres2. In order to improve the volumetric hydrogen storage capacity, solid-state storage in metal hydrides is considered as an effective approach8. As an example, excluding the volume of the tank material, 5 kg of hydrogen can be stored in magnesium hydride (MgH2) occupying a volume of only 46 litres7. However, due to its high desorption enthalpy (ΔHdes = 74 kJ mol−1), MgH2 requires high dehydrogenation temperatures (>300 °C)9. By the reaction of alkali metals  with gaseous ammonia, metal amides (i.e. NaNH2 and KNH2) were firstly discovered at the beginning of 1800s11. During the last 50 years, metal amides were not considered as potential hydrogen storage materials since the detected main gaseous product from their thermal decomposition was ammonia13. In 2002, Chen et al. reported for the first time that a material composed of LiNH2 and LiH was able to reversibly store 6.5 wt. % of H2 at 255 °C14. Thereafter, a focus was given to synthesize and understand the mechanisms in amide-imide systems for hydrogen storage16. Replacing LiNH2 with Mg(NH2)2, a reversible H2 storage capacity of 5.5 wt. % at operating temperatures of 200 °C is obtained according to reactions (1) and (2) (ΔHdes = 40 kJ mol−1)17.One of the limiting factors for the implementation of hydrogen in stationary and mobile applications is the lack of an efficient and safe storage system. For mobile applications, a fuel cell equipped electrical car requires about 5 kg of hydrogen in order to achieve a driving range of ca. 500 km2)2 + 2LiH stoichiometric mixture, operating temperatures of 90 °C can be achieved at 1 bar18. However, sufficient dehydrogenation rates, even after intense ball milling treatment, can be obtained only at temperatures above 180 °C due to harsh kinetic constraints19. Several attempts have been made in order to improve the sluggish dehydrogenation behaviour of the Mg(NH2)2 + 2LiH composite system39. Potassium containing additives effectively reduce the dehydrogenation peak temperature down to 130 °C, which is ∼50 °C lower than that of pristine Mg(NH2)2 + 2LiH42. However, due to segregation phenomena that occurs at high-temperature (≥180 °C) upon cycling, the inhomogeneous distribution of the K-species reduces their catalytic activity43. Therefore, the design/synthesis of new additives is mandatory in order to achieve long-lasting absorption/desorption properties. TiO2 is one of the low-cost additives which enhance the hydrogen storage properties of the 2LiBH4 + MgH2 reactive hydride composite (RHC) system46. Puszkiel et al. showed that 2LiH + MgB2/2LiBH4 + MgH2 RHC system doped with core-shell LixTiO2 nanoparticles shows improved the kinetic and cycling behaviour44. It was found that the core-shell LixTiO2 nanoparticles act as Li+ pumps, increasing Li+ mobility, hence accounting for the observed enhanced hydrogen storage properties. Studies on reaction mechanism of Mg(NH2)2 + LiH system showed that diffusion of small ions  might account for the improved reaction kinetics50. In this work, we investigate the effect of LixTiyOz and potassium-modified LixTiyOz additives on the microstructural and hydrogen storage properties of Mg(NH2)2 + 2LiH system.According to the calculated thermodynamic properties of the Mg(NH2)2 (described in the following subsection), LiH , anatase TiO2  and KH . The investigated additives were obtained by milling LiH, TiO2 and KH in different stoichiometric ratios under argon atmosphere for two hours and then annealing them under Ar atmosphere at 600 °C for 8 hours. The stoichiometry of the reagent utilized to synthesize the additives were: 1) 0.5LiH + TiO2 and 2) 0.5LiH + TiO2 + 0.25KH. In addition to the prepared additives, KH alone was also used as an additive. In order to separate mineral oil from KH, three washing cycles in hexane were carried out. After that, hexane was removed by applying dynamic vacuum.All reagents utilized in this work were Mg(NH2)2 (95 % purity) was in-house synthesized by ball milling MgH2 under NH3 atmosphere, followed by annealing at 300 °C under NH3 atmosphere. The details of the synthesis were described in our previous study50. The Mg(NH2)2 was mixed with LiH  and 1.0, 2.5 or 5 mol. % of additives (Section 2.2). All materials were milled in a Fritsch P6 Planetary ball miller for 5 hours with ball to powder ratio 60:1 under 50 bar of H2 pressure. The sample names used to identify the prepared specimens are listed in Table Mg(NHEx situ powder X-ray diffraction method (PXD) was applied for the identification of crystalline phases, by using a Bruker D8 Discover diffractometer equipped with Cu X-ray source (λ = 1.54 Å) operating at 50 kV and 1000 mA and a 2D VANTEC detector. Diffraction patterns were collected in the 2θ range 20° to 80°. A sample holder sealed with a polymethylmethacrylate (PMMA) dome was utilized to prevent the material oxidation during PXD measurements.In situ synchrotron radiation powder X-ray diffraction (SR-PXD) technique was applied using a special designed cell51. This cell with sapphire capillary allows performing measurements under controlled gas atmosphere in a pressure range from 0.01 to 200 bar. Measurements were performed at Deutsches Elektronen-Synchrotron (DESY) in the P02.1 beamline. The beamline is equipped with A Perkin Elmer XRD1621 area detector and 60 keV X-ray source (λ = 0.207 Å). Mg-Li-5LTOK sample was heated from room temperature (RT) to 300 °C with a heating rate of 5 °C/min under 1 bar of H2 pressure. Every 10 seconds a two-dimensional SR-PXD pattern was collected. Collected data were integrated to one-dimensional diffraction pattern using Fit2D software53.2O and O2 levels below 1 ppm). Before starting the DSC measurements, the residual argon gas inside the chamber was removed by first evacuation and then flushing the chamber with hydrogen. A mass flow-meter was used to limit the deviation of the hydrogen pressure to ±0.2 bar of H2 during heating up and cooling down. In order to measure apparent activation energies of the 1st and 2nd desorption, about 10–15 mg of each sample were placed in a Al2O3 crucible and then heated from room temperature (RT) up to 300 °C under 1 bar of H2 pressure with heating rates of 1 °C, 3 °C, 5 °C and 10 °C/min. For the 2nd desorption, as-milled samples were first desorbed by heating from RT to 220 °C under 1 bar of H2 pressure. Following this step, the materials were reabsorbed by heating them from RT to 180 °C under 100 bar of H2 pressure.Differential scanning calorimetry (DSC) measurements were performed in a Netsch DSC 204 HP calorimeter located inside an argon-filled glovebox (HEa) of the 1st and 2nd desorption reactions were calculated via Kissinger method54. This method is suitable for the samples that exhibit multi-step reactions and it allows us to determine Ea of a reaction process without assuming a specific kinetic model, i.e. without determining the rate-limiting step of the reaction. The equation for the Ea calculation is shown in Eq. A is the pre-exponential factor and R is the gas constant. The temperature for the maximum reaction rate (Tm) was obtained from DSC curves measured at measured heating rates (β) of 1 °C, 3 °C, 5 °C and 10 °C/min. Then, m was plotted, Ea (kJ/mol H2) and A (1/s) was calculated from linear fitting. Goodness of fit was determined by the examining the correlation between the experimental and predicted values. In order to have a good fitting, R-square value should be near 150.In order to evaluate the effectiveness of the additives on the material kinetic behaviour, the apparent activation energies  is taken as the hydrogen capacity over the maximum reached capacity for each sample. By implementing different rate equations, several plots of st, 2nd and 5th absorption/desorption curves between 0.1 and 0.8 fractions of the overall hydrogen capacity. The best fitting reaction rate model must obey the following rules; slope of the fitted line should be ∼1, intercept ∼0 and R2 ∼ 1. Details related to the implemented rate equations are given in our previous work50.In order to assess the rate-limiting steps of the absorption/desorption processes in the studied system, Sharp and Jones method was applied2O and O2 levels below 1 ppm). Each measurement was acquired in the transmission mode in spectral range of 650 cm−1–4000 cm−1 with a resolution of 4 cm−1 50.IR spectroscopy was performed with an Agilent Technologies Cary 630 FT-IR located in an argon filled glove box . About 2 mg of sample was placed in a Al2). The temperature and pressure conditions are provided in the figure caption for each experiment in the manuscript. The mass of sample for all the measurements was approximately 100 mg.The absorption rates and gravimetric capacities were assessed using a Sieverts apparatus  operating on the differential pressure technique. The hydrogen gas used in the experiments had a purity of 99.999 % (5.0 H2 and H2O atmosphere (<1 ppm) by dispersing the powders onto carbon grids. In order to avoid the oxidation/hydrolysis of the material at the time to introduce the grids into the microscope column, the dispersed powder on the grid was covered with a special polymeric film which does not preclude the electron interactions with the sample57. Then, HR-TEM observations of the identified Fe zones were done. HR-TEM image processing was done with the following programs: Digital Micrograph (License no. 90294175), i-TEM .High resolution transmission electron microscopy (HR-TEM) observations, diffraction patterns (DP) and dark field (DF) were carried out using a Tecnai G2 microscope with an information limit of 0.12 nm and Schottky Emission gun operating at 300 kV. Samples after milling and after absorption/desorption conditions were observed. All samples were prepared into a glove box with controlled O2O4, K-modified additive , TiO2 anatase, as as-milled Mg-Li-5LTO, as-milled Mg-Li-5LTOK, as-milled Mg-Li-2.5LTOK, desorbed Mg-Li-2.5LTOK and reabsorbed Mg-Li-2.5LTOK samples were carried out using a R-XAS looper “in house” spectrometer from Rigaku. The measurements were performed in transmission mode around the Ti K-edge (4966 eV) in the range of energy from 4950 eV to 5030 eV at ambient temperature. The optimum amount of material for the measurements was calculated by the program XAFSMAS 58. The samples were prepared inside a glove box by mixing them with anhydrous boron nitride  in a mortar, and then pressing the mixture into pellets of 10 mm diameter. The pellets were sealed with Kapton tape (50 μm in thickness) to prevent the oxidation/hydrolysis of the samples. XAS data processing and fitting were performed by using the IFEFFIT software  package59.X-ray absorption spectroscopy experiments at the XANES (X-ray absorption near edge structure) region of LiTiex situ PXD, in situ SR-PXD and infrared spectroscopy for all the compositions are presented in this section. In Table 2 stoichiometric composition indicates that the additive is just composed of the LiTi2O4 after milling and annealing  evolving from the two samples upon heat treatment are almost identical . However, due to the broadness of the observed diffraction peaks, we cannot exclude the presence of several phases having a general formula LixTiyO4 . This fact suggests that the additive´s composition changes upon milling. The presence of reflections belonging to Li2Mg(NH)2 (orthorhombic phase) at around 170 °C indicates that the desorption reaction has already started, which is in good agreement with DTA analysis 2 takes place at the temperatures higher than 220 °C. This transition is expected since the phase transformation of Li2MgN(H)2 from the orthorhombic to the cubic structure occurs over 200 °C60. Unfortunately, in this analysis, it was not possible to identify any crystalline potassium compounds. This implies that potassium-containing phases are either in amorphous or nanocrystalline state.The thermal behaviour of as-prepared samples is presented in Fig. cal Fig. . HoweverPXD Fig. . The PXDsis Fig. . The for2)2 + LiH system42. In order to compare our findings with the pure KH added system, Mg-Li-5K sample (Mg(NH2)2 + 2LiH + 0.05KH) was prepared. Despite the fact that the lowest onset temperature (135 °C) is obtained with this sample, its reaction rate is slower in comparison with the samples containing K-modified additive.First desorption kinetics of as-milled samples are presented in Fig. 2 notably faster . Therefore, the effect of K-modified additive on Mg-Li is clearly seen both in the absorption and desorption kinetic properties. Despite the fact that Mg-Li-1LTOK has fast H2 absorption kinetic, the H2 capacity is reduced from 4.3 to 3 wt. % after a single cycle. After cycling, the best sample that has a good H2 absorption kinetic and cycling stability is Mg-Li-2.5LTOK. For this reason, we chose this sample to further investigate its cycling stability compared to Mg(NH2)2 + 2LiH system.Reabsorption kinetics of Mg-Li and Mg-Li-5LTO are sluggish and require more than 10 hours to reach full capacity Fig. . On the Ea), the Kissinger method54 was applied for the 1st and 2nd desorption reactions of the Mg-Li, Mg-Li-5LTO, Mg-Li-5LTOK and Mg-Li-2.5LTOK samples. For the calculations, the peak maximum of the main thermal event . On the contrary, Ea value rises to 211 ± 1 kJ/mol H2 for the sample Mg-Li-2.5LTOK. It is worthy to note that frequency factor of this sample is considerably higher (A = 1.2 × 1019 s−1) compared to the ones of the Mg-Li, Mg-Li-5LTO and Mg-Li-5LTOK samples.In order to evaluate the effect of the modified additives on desorption apparent activation energy 2 + 2LiH hydride system, i.e. reduced desorption temperature, fast reabsorption kinetic. Mg-Li-2.5LTOK sample exhibited the highest reversible H2 storage capacity of about 3.5 wt. %  space group and a broad peak at 2θ = 30°, which corresponds to the tetragonal Mg(NH2)2 structure with I41/acd(142) space group. Since Mg(NH2)2 is amorphous after intense ball milling, it can be hardly observed in PXD61. In contrast, it is more visible on the FT-IR pattern 2 are positioned at 3268 and 3324 cm−1. When Mg-Li is half desorbed, LiNH2 can be detected at 3257 and 3310 cm−1 263. PXD reflections coming from the cubic Li2Mg(NH)2 phase with iba2(45) space group are found in the half and fully desorbed samples 2 2 Fig. .Figure 5xTiyO4 compounds with 0.75 ≤ x ≤ 1 and 1.9 ≤ y ≤ 2 are present. In the ICSD database, it is possible to find several crystal structures belonging to LixTiyO4 that fit well with all reflections64. These formed phases are stable and their peaks positions do not change within desorption/absorption processes. The compositions of the as-synthesized additives were already presented in the introduction of the results section 2 and LiH leads to some changes in the additives´ composition, which will be later discussed in the following section.Regarding the additives, PXD analyses Fig.  reveal txTiyOz and K-modified LixTiyOz additives on the Mg(NH2)2 + 2LiH system were studied. K-modified additive not only plays a role on improving the reaction kinetic behaviour 2 and LiH changes. XRD analyses of the as-milled materials . Nevertheless, the composition of the additives in the LTOK after milling is not clear yet. Therefore, X-ray absorption spectroscopy near edge structure (XANES) technique was applied to Mg-Li-5LTO, Mg-Li-5LTOK and Mg-Li-2.5LTOK samples in order to investigate the oxidation state of Ti. The changes in the oxidation state of Ti were determined by the shift of the absorption edges of the samples. The results were compared with the measured XANES spectra of TiO2 and LiTi2O4 reference materials. In Fig. 2O4: 100 %, ESI Fig. 1.04O16Ti8, 17 wt.% of LiTi2O4, 27 wt.% LiTiO2 and 46 wt.% K2O17Ti8, ESI Fig. et al. already reported that Ti in LiTi2O4 has an effective valence state of +3.565. Thus, the effective oxidation state of Ti in LTO and LTOK additives is +3.5. Comparing the XANES spectra of the as-milled Mg-Li-5LTO, the LTO additive and anatase TiO2  as shown in Fig. 2O4 (Ti+3.5) and 76 % of other species, suggesting that the effective Ti valence state is slightly smaller than the presented by the Mg-Li-2.5LTOK sample.As we reported in Fig. als Fig.  providedESI Fig. , it is pESI Fig. , with a ESI Fig. . Then, t2)2 compound. Due to the thickness of the rings, it is not possible to discard some intermediate (LiNH2), product (Li2Mg(NH)2) and by-product (Li3N and Mg3N2) species. However, these phases are not expected in the as-milled state, unless Mg(NH2)2 and LiH interacts during the observations prompted by the energy of the beam. It is also possible to attribute the observed reflections to species composed of Li-Ti-O  and LiTi2O4 (cubic) are confirmed by the structure analyses of the HR-TEM photos. For the as-milled Mg-Li-2.5LTOK  and LiTi2O4 (cubic) are found. Based on the position of the absorption edge of the Mg-Li-2.5LTOK sample compared to the ones from references TiO2 and LiTi2O4  and crystal structure simulation analyses were performed. Figure TOK Fig. , nanopar2O4 Fig. , we can Ea for the first and second desorption reactions  was calculated by the Arrhenius expression k = A · exp[−Ea/RT] (1/s) at 180 °C, which is the cycling temperature 2 and later metathesizes with LiH to regenerate KH41. Based on our results from in situ SR-PXD contour plot 2 + 2LiH system due to high electronegativity of K (0.82 eV), Ti (1.54 eV) and O (3.44 eV) elements. Therefore its acts as a catalyst rather than changing the thermodynamics of the system.Therefore, these outcomes suggest that the presence of the Klot Fig.  togetherlot Fig. , we propxTiyOz and K-modified LixTiyOz additives on the Mg(NH2)2 + 2LiH system were studied. 5 mol. % additive containing sample Mg-Li-5LTOK reduced the desorption peak temperature of pristine sample by 30 °C and suppressed NH3 release until 220 °C. Although Mg-Li-2.5LTOK has comparably higher apparent activation energy (211 ± 1 kJ/mol) respect to Mg-Li (183 ± 7 kJ/mol), calculated rate constant (k) value was bigger during the first and second desorption reactions which is in agreement with reaction behaviour. Orthorhombic K2TiO3 and cubic LiTi2O4 phases were detected in HR-TEM observations, where oxidation state of Ti was in accordance with XANES analysis. Based on our results from in situ SR-PXD plot and XANES analysis, we propose that K2TiO3 nanoparticles act as catalyst and they positively affect the reversible reactions of the Mg(NH2)2 + 2LiH system due to high electronegativity of K (0.82 eV), Ti (1.54 eV) and O (3.44 eV) elements.In this work, microstructural and kinetic effects of LiImproved kinetic behavior of Mg(NH2)2-2LiH doped with nanostructured K-modified-LixTiyOz for hydrogen storage


Antioxidant assays showed that MPPs had strong antioxidant activities, with 92 ± 4.2% of DPPH radical scavenging rate, 79 ± 2.5% of ABTS radical inhibition rate and 4.7 ± 0.5 μM Trolox equivalents per kg−1 DMP of ferric reducing power. Gallic acid possess a stronger antioxidant capacity than other phenols. In vitro cytotoxic tests suggested that mango peel extract (MPE) had an IC50 value of 15 mg·mL−1 and MPPs had a stronger inhibitory effect on the A549 cell line. Oleanolic acid exhibited the strongest cytotoxicity, with an IC50 value of 4.7 μM, which was similar with that of the positive control 5-fluorouracil.Mango peel, the main by-product of juice processing, possesses appreciable quantities of bioactive phenolic compounds and is worthy of further utilization. The present work reports for the first time the HPLC analysis and in vitro antioxidant evaluation of mango peel phenols (MPPs) and their cytotoxic effect on the A549 lung cancer cell line. These results indicated that mango peel has the total phenolic content of 723.2 ± 0.93 mg·kg Mangifera altissima, M. applanata, M. indica, etc., which M. indica L. is one of the most cultivated varieties. As of 2005, China has been listed as the second largest producer of mango, with 12.9% of the world's output. Mango fruit is suitable for not only fresh eating, but also processing into juices, chutneys, sauces and preserved fruit. Worldwide this accounts for about 25% of mango production. However, the large amount of by-products, like mango skins mango seeds , causes environmental pollution [M. indica) peel is a rich source of phenolic compounds, with amounts up to 0.55~1.1 g·kg−1 dry mango peel (DMP) [M. indica L.) peel extract (MPE) and its MPPs, including DPPH•, ABTS•+ and FRAP scavenging, as well as their cytotoxic effect on the A549 human lung carcinoma cell line.Mango is one of the most popular fruits, with high nutrition and commodity value, which is grown in over 70 countries in the world and includes about 35 species, including el (DMP) ,7,8,9, f−1 DMP in MPE of M. indica L. As shown in A), caffeic acid (B), chlorogenic acid (D), gallic acid (E), procyanidin B2 (F) together with oleanolic acid (C) (−1) followed by procyanidin B2 (12.5 ± 0.61 mg·kg−1), caffeic acid (11.4 ± 0.08 mg·kg−1), gallic acid (7.0 ± 0.57 mg·kg−1), oleanolic acid (3.0 ± 0.25 mg·kg−1), vanillic aldehyde (0.55 ± 0.04 mg·kg−1).Using the modified Folin–Ciocalteu method, the total phenolic content was determined to be 723.2 ± 0.93 mg·kgacid (C) . Quantit–1, which were 92 ± 4.2%, 79 ± 2.5%, 4.7 ± 0.53 μmol TE·kg–1 DMP, respectively. In order to identify the potential contributor(s) in MPE, six detected phenolic compounds were further evaluated their antioxidant effects. As shown in –1 DMP), which was consistent with the findings revealed by Yen and his coworkers [−1 DMP of reducing power.An in vitro antioxidant assay indicated that MPE had strong capacity of scavenging DPPH and ABTS radicals and ferric reducing power at 2.0 mg·mLoworkers . The ant50 value of 15 mg·mL−1, while caffeic acid, oleanolic acid, chlorogenic acid and procyanidin B2 had potent inhibitory effects. Among these MPPs, oleanolic acid had the strongest cytotoxic effect, with an IC50 value of 4.7 μM, which was similar to that of the positive control 5-fluorouracil (3.8 μM).As shown in These findings were consistent with the morphological variations of the nucleus of A549 cells observed using 4′,6-diamidino-2-phenylindole (DAPI) staining. As shown in M. indica L.) was purchased from a fruit market in Hangzhou (China). After peeling, lyophilizing and grinding, about 20 g of DMP powder was extracted with 200 mL of 70% aqueous ethanol (50 °C) under stirring for 20 min using microwave-assisted extraction . The obtained MPE was concentrated under reduced pressure and lyophilized . After redissolving in 500 mL of 20% ethanol solution, the resulting extract was centrifuged for 15 min at 60,000× g  and retained on a normal atmosphere column with 200 mL macroporous absorbent resin XAD-16  followed by eluting with three bed volumes of 80% alcohol in H2O. The collected elution was concentrated under reduced pressure and lyophilised. The afforded phenol-enriched extract was dissolved in methanol with a final concentration of 20 mg·mL−1 and preserved at 4 °C before HPLC analysis. The total phenolic content of MPE was determined by the modified Folin–Ciocalteu method proposed by Wijngaard and Brunton [−1 DMP).Fresh mango  solution were mixed with 2 mL of 2,2-diphenyl-1-picrylhydrazyl radicals . The reaction mixture was shaken vigorously and stored in the dark for 30 min at room temperature. The absorbance of the solution was then recorded on a Hitachi-UV-3000 spectrophotometer  at 517 nm. 100 µL of methanol was added to the DPPH solution used as a blank control. All measurements were performed in triplicate. The percentage scavenging effect was calculated as follows:IDPPH is the DPPH radical scavenging rate (%), A0 is the absorbance of a negative control, A1 is the absorbance of each sample [MPP (100 µL) or oleanolic acid (100 µM) or MPE (2.0 mg·mLh sample .IABTS is the ABTS•+ inhibition rate (%), A0 is the absorbance of a negative control, A1 is the absorbance of each chemical (100 µM) or MPE (2.0 mg·mL-1) solution.The capability of scavenging 2,2′-azinobis radical was carried out according to the method described by Bao and his co-workers . The ABT−1) solution or methanol (negative control) in 1 cm disposable plastic cells and incubated at 37 °C for 120 min. The absorbance was taken on a Hitachi-UV-3000 spectrophotometer at 595 nm. A standard curve was obtained using Trolox solution at 0.5, 1.0, 1.5, 2.0 mM. The absorbance of each sample was compared with that of the Trolox standard and the results were expressed in terms of micromole Trolox equivalents per kg DMP (µM TE·kg−1 DMP). All measurements were performed in triplicate. The ferric reducing power was calculated by the following equation:IFRAP is the ferric reducing power, A0 is the absorbance of a negative control, A1 is the absorbance in the presence of a tested sample [Fresh FRAP reagent was prepared by mixing 2.5 mL of tripyridine triazine , 2.5 mL of ferric chloride (20 mM) and 25 mL of sodium acetate buffer . 900 µL of the FRAP reagent were mixed with 300 µL of each phenol (100 µM) or MPE (2.0 mg·mLd sample .−1) penicillin-streptomycin  at 37 °C in a humidified 5% CO2 incubator . The medium was changed daily and subcultured when they reached a confluence of 80%.Human NSCLC cell lines A549 were obtained from the Cancer Institute of Sun Yat-Sen University  and cultured in RPMI-1640  medium containing 10% FBS  and 1%  culture resulting in final concentrations of 6.25, 12.5, 25, 50, 75, 100, 150, 200 μM, respectively. MPPs and oleanolic acid were also dissolved in accordance with the above methods in final concentrations of 68.5, 100, 200, 300, 400, and 500 μg·mL−1, with 5-fluorouracil as the positive control.Six chemicals including vanillic aldehyde, caffeic acid, oleanolic acid, chlorogenic acid, gallic acid and anthocyanidin B3 per well were incubated for 24 h at 37 °C. Then the supernatant was removed and the cells were treated for 24 h with chemicals at different concentrations  or MPPs at different concentrations . CCK-8 (10 μL) dissolved in PBS (5 mg·mL−1) was added to each well, and the cells were further incubated for 45 min. Finally, the medium containing CCK-8 was removed and 100 μL of DMSO was added to each well. The plate was gently shaken for 10 min to dissolve the formazan crystals  and the absorbance was measured at 450 nm on a ELISA . The percentage viability was calculated using the following formula:The effect of MPPs and oleanolic acid on cell proliferation of A549 was evaluated by cell counting kit-8 (CCK-8) assay according to the manufacturer’s instruction. Briefly, cells in 96-well plates at a density of 5 × 1050) was calculated from the dose response curves for each cell line [Sample group , blank group (90 µL cell medium + 10 µL sample solution + 10 µL CCK-8 reagent), negative groups (90 µL cell suspension + 10 µL cell medium + 10 µL CCK-8 reagent). The concentration of MPPs and oleanolic acid needed to inhibit cell growth by 50%  1 mL and MPPs and oleanolic acid (100 μg·mL−1), 5-fluorouracil  as the positive control group and blank group, added 1 mL cultures and incubated at 37 °C for 48 h [−1) (Sigma-Aldrich) and kept in dark at 37 °C for 30 min, after the incubation, washing with buffer, add 1 mL culture, in the inverted fluorescence microscope observed under fluorescent green fluorescence set  and take photos, and use the Image-ProPlus 5.1 software  processing images, green fluorescence intensity ratio calculation [The logarithmic phase of A549 cells, using 0.25% of trypsin digestion, counting, add broth, made 5×10for 48 h ,18. The culation ,20.p < 0.05 were considered significant.All data were expressed as mean ± standard deviation (SD) and analyzed by SPSS software . Values of 2 and oleanolic acid. Antioxidant assays suggested that MPE had strong DPPH and ABTS radical scavenging capacity and ferric reducing power at 2.0 mg·mL−1. Gallic acid possessed the strongest DPPH• scavenging rate (97 ± 0.4%), ABTS•+ inhibition rate (86 ± 3.0%) and reducing power (12 ± 0.5) µmol TE·kg−1 DMP. In vitro CCK-8 assay indicated that oleanolic acid exhibited the strongest inhibitory effect on A549 cell line with an IC50 value of (4.71 ± 0.30 μM). Fluorescence staining experiments confirmed that oleanolic acid inhibited cell proliferation by destroying the nucleus of A549 cell. These findings would promote the utilization of mango peel and the development of antioxidant food.The present work firstly reported on in vitro antioxidant activities of phenols and oleanolic acid from mango peel and their cytotoxic effect on the A549 cell line. The ethanolic extract of mango peel is rich in phenolic compounds. HPLC analysis indicated that MPE had five main chemical constituents, including vanillic aldehyde, caffeic acid, chlorogenic acid, gallic acid, procyanidin B


In order to prepare 4-[18F]fluorophenol in no-carrier-added (n.c.a.) form only a nucleophilic labelling method starting from [18F]fluoride is suitable. In this paper a new, two step radiosynthesis starting from 4-benzyloxyphenyl-(2-thienyl)iodonium bromide and [18F]fluoride with subsequent deprotection is described, yielding n.c.a. [18F]fluorophenol in 34 to 36% radiochemical yield.4-[ Especially, the introduction of no-carrier-added (n.c.a.) [18F]fluoride into an aromatic ring is difficult, especially if it is not activated for a nucleophilic substitution reaction fluorobenzenes can easily be formed as intermediates and subsequently transformed by as few as possible steps to the desired radiotracer.The introduction of fluorine into organic molecules plays an important role in the development of new as well as in routine production of established pharmaceuticals . The usereaction . In thos18F-labelled intermediate is n.c.a. 4-[18F]fluorophenol, which has proven its suitability in various radiosyntheses of biological relevant molecules or potential radiopharmaceuticals [18F]fluorophenol [18F-labelling precursors even for electron rich arenes [18F]fluorophenol.A further versatile primary euticals ,4. Durinrophenol ,6. The orophenol  which ar18F]fluoride excludes a direct 18F-introduction into an unprotected phenol-substituted iodonium compound. Since the benzyl protective group offers an easy and gentle way of deprotection, it was chosen for this purpose. Thus, 4-benzyloxyphenyl-(2-thienyl)iodonium bromide (1c) was examined as precursor. This precursor was prepared staring from 4-benzyloxy-1-iodobenzene (1a), which was oxidized by sodium periodate to 4-benzyloxy-1-(diacetoxyiodo)benzene (1b) within 2 h with a yield of 66%, as described in the literature 1dfluorophenol 18F]1fluorophenol under the same conditions. By filtration of the reaction suspension via a LiChrolut© glass column containing a PTFE frit, n.c.a. 4-[18F]fluorophenol was obtained in an anhydrous methanol system. A flow chart of the radiosynthesis is depicted in For subsequent removal of the benzyl-moiety a reductive deprotection, applying ammonium formate and palladium black as catalyst in methanol, was elaborated. First attempts with ammonium formate and palladium on activated charcoal led to the desired n.c.a. 4-[18F]fluorophenol, described by Ludwig et al. fluorophenol is obtained in an anhydrous methanol system which allows for direct further radiosynthetic steps including compounds susceptible to moisture without time-consuming drying procedures. On the other hand, an unfavourable aspect is the overall lower RCY of 35 ± 1% compared to 55% with the benzophenone method. This, however, is in part compensated by the shorter synthesis time.In comparison to the so far best alternative procedure for the preparation of n.c.a. 4-fluoride is routinely carried out at the JSW BC 1710 Cyclotron (Forschungszentrum Jülich) by bombardment of a 96% isotopically enriched [18O]water target  with a 17 MeV proton beam using the 18O18F nuclear reaction [18F]fluoride was separated from the enriched [18O]water by an electrochemical cell [18F]fluoride can be produced within 1 h at a nominal beam current of 25 μA.The production of n.c.a. fluoride solution 30 to 50 MBq (10–50 μL) were added to Kryptofix© 2.2.2 (26 μmol) and a 1 M potassium carbonate solution (13 μL). After adding anhydrous acetonitrile (0.8 mL) the mixture was transferred through the septum into the prepared vial by a 1 mL syringe. For azeotropic distillation the vial was heated in an oil bath to 82 °C under a constant flow of argon adjusted at 750–850 mbar until no acetonitrile/water mixture had been left. This step was repeated by adding acetonitrile (1 mL × two times) which was subsequently followed by 5 min of full vacuum (~10 mbar). After argon flushing and pressure balancing the resulting dry n.c.a. [18F]fluoride-cryptate complex was ready for the nucleophilic 18F-labelling reaction.A 5 mL Wheaton18F]fluoride-cryptate complex and then stirred under 1,100 mbar argon at 130 °C. At appropriate times aliquots (10 μL) were taken with a micro-syringe (Hamilton©) via the septum and added to cold acetonitrile (50 μL). These samples were analyzed by radio-HPLC and/or radio-TLC. After 15 to 20 min the whole reaction mixture was added into water (10 mL) using a 20 mL syringe. This solution was passed through a SepPak© C18 plus cartridge preconditioned with ethanol (3 mL) and water (5 mL). The cartridge was washed with water (3 mL) and dried by an argon flow for 5 min. Before elution, the cartridge was connected to a dry SepPak© ALOX cartridge. The product absorbed on the C18 phase was eluted with methanol (3 mL) and transferred through both cartridges into a new reaction vial. 4-Benzyloxy-[18F]fluorobenzene (18F]1d and radio-TLC . Ammonium formate (~5 mmol) and palladium black (~0.15 mmol) were added to the methanol solution. For debenzylation the mixture was stirred at 80 °C for 15–20 min. The whole reaction mixture was filtered through a Merck LiChrolut© glass column (65 × 10 mm) containing only a PTFE frit (porosity 10 μm) which was washed with 0.5 mL of methanol. The obtained n.c.a. 4-[18F]fluorophenol was analyzed by radio-TLC  and radio-HPLC ([k’ = 1.93] acetonitrile/water (40:60), flow 0.7 mL/min).4-Benzyloxyphenyl(2-thienyl)iodonium bromide (25 μmol) was dissolved in anhydrous DMF (1.0 mL) and added by a syringe through the silicone septum into the vial containing the dry n.c.a. [18F]fluorophenol from 4-benzyloxyphenyl(2-thienyl)iodonium bromide (1c) presents an improved radiosynthesis of this primary 18F-labelled synthon, especially in terms of practicability of labelling and applicability of coupling chemistry in subsequently radiosynthesis steps. Thus, n.c.a. 4-[18F]fluorophenol was synthesised by nucleophilic substitution on 4-benzyloxyphenyl(2-thienyl)iodonium bromide and subsequent debenzylation within 40 min and an overall RCY of 34% to 36%. This new approach towards the versatile 18F-labelling synthon 4-[18F]fluorophenol offers a short and very convenient two synthetic and one purification step procedure. In addition, the final 4-[18F]fluorophenol is obtained in an anhydrous methanol solution which facilitates a broad variety of following coupling steps, even with moisture sensitive molecules. Preparation of the useful 4-[18F]Fluorophenol via the 4-benzyloxyphenyl(2-thienyl)iodonium bromide precursor thus requires a far less complex radiosynthesis which enables an easy transfer to a remotely controlled synthesis device.In conclusion, the reported preparation of n.c.a. 4-[


Dear Editor,Several recent clinical studies have indicated that dietary supplementation with branched-chain amino acids (BCAA), particularly with leucine, is an effective anti-atrophic therapy  muscle mass decreased to 70.9% ± 1.8% of the contralateral level, while GCN2-deficient TA muscle mass remained at 83.1% ± 1.6% of its contralateral level . After denervation for ten days, the diameter of WT FDB myofiber decreased from 37.2 ± 0.9 μm to 24.2 ± 0.7 μm, while the diameter of Gcn2−/− FDB myofiber was maintained at 32.3 ± 0.7 μm. Overexpression of GCN2 resulted in a further reduction in the diameter of FDB myofibers in both WT and Gcn2−/− mice. However, the reduction was more dramatically in Gcn2−/− FDB muscle and the significant difference in the diameter of atrophied FDB myofiber between WT and Gcn2−/− mice was diminished after transfected with the GCN2 plasmid  and Atrogin-1, MuRF-1 and LC3 .Emerging evidence suggests that the protein degradation in muscle atrophy is mediated by FoxO3a, an important regulator of To investigate whether GCN2 activation directly causes FoxO3a nuclear translocation in muscle atrophy, we generated a stable C2C12 cell line (mGCN2-C2C12) with doxycycline (Dox)-controlled expression of flag-tagged mouse GCN2  and Chinese Academy of Sciences .Yuting Guo, Huiwen Wang, Yinglong Tang, Yue Wang, Mengqi Zhang, Zhiguang Yang, Eric Nyirimigabo, Bin Wei, Zhongbing Lu, and Guangju Ji declare that they have no conflicts of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.Supplementary material 1 (PDF 474 kb)Below is the link to the electronic supplementary material.


Enterobacter aerogenes strains are a major clinical problem because of the lack of effective alternative antibiotics. However, viruses that lyze bacteria, called bacteriophages, have potential therapeutic applications in the control of antibiotic-resistant bacteria. In the present study, a lytic bacteriophage specific for E. aerogenes isolates, designated vB_EaeM_φEap-3, was characterized. Based on transmission electron microscopy analysis, phage vB_EaeM_φEap-3 was classified as a member of the family Myoviridae . Host range determination revealed that vB_EaeM_φEap-3 lyzed 18 of the 28 E. aerogenes strains tested, while a one-step growth curve showed a short latent period and a moderate burst size. The stability of vB_EaeM_φEap-3 at various temperatures and pH levels was also examined. Genomic sequencing and bioinformatics analysis revealed that vB_EaeM_φEap-3 has a 175,814-bp double-stranded DNA genome that does not contain any genes considered undesirable for the development of therapeutics . The phage genome contained 278 putative protein-coding genes and one tRNA gene, tRNA-Met (AUG). Phylogenetic analysis based on large terminase subunit and major capsid protein sequences suggested that vB_EaeM_φEap-3 belongs to novel genus “Kp15 virus” within the T4-like virus subfamily. Based on host range, genomic, and physiological parameters, we propose that phage vB_EaeM_φEap-3 is a suitable candidate for phage therapy applications.Carbapenem-resistant  Enterobacter aerogenes has increasingly been recognized as an important opportunistic and multidrug-resistant bacterial pathogen associated with nosocomial infections  was added and mixed, and overlaid on the solidified base nutrient agar (1.5% agar). Following incubation overnight at 37°C, clear phage plaques were picked from the plate. A pure phage suspension was obtained by three rounds of single-plaque purification and reinfection of the exponentially growing 3-SP strain, as reported previously  to a concentration of ∼109 PFU/mL. Samples were processed by negative staining with 2% (wt/vol) uranyl acetate for 30 s and examined using a Tecnai Spirit 120-kV transmission electron microscope  at different magnitudes to determine the phage morphology.The phage particle preparation was centrifuged at 20,000 ×E. aerogenes strains, 16 enterobacterial isolates , three Gram-negative non-fermenters , and one Gram-positive bacterium . Each strain was grown in 5 mL of Luria–Bertani (LB) broth at 37°C to an optical density at 600 nm (OD600) of 0.5 before being centrifuged at 4000 × g for 5 min and resuspended in 3 mL of SM buffer. A 0.2-mL aliquot of bacterial suspension was then mixed with 3 mL of molten soft-agar and poured onto an LB agar plate. After the agar had solidified, 0.01 mL of phage suspension was spotted onto the overlay. Sensitivity of the bacterium to vB_EaeM_φEap-3 infection was assessed following overnight incubation as described previously (The host range of phage vB_EaeM_φEap-3 was determined via the spot test method using 28 eviously .600 = 0.4–0.5) before being centrifuged at 4000 ×g for 10 min at 4°C. The cell pellet was then resuspended in a 0.1-volume of SM buffer. A 0.1-mL aliquot of phage suspension was then added to 0.9 mL of the bacterial suspension to achieve a multiplicity of infection of 0.01. Phages were allowed to absorb for 5 min at 37°C, and then the mixture was centrifuged twice at 16,000 ×g for 2 min to remove the unabsorbed phages. The mixtures were then resuspended in 10 mL of LB broth and incubated at 37°C. Samples were collected at 10-min intervals for 80 min with or without 1% chloroform and immediately diluted and plated for phage titer assays  at 4, 25, 37, 50, 60, 70, or 80°C for 15, 30, 45, or 60 min. After treatment, tubes were cooled and serial dilutions of each sample were tested against strain 3-SP in a double-layer agar assay to measure the lytic activity of the phage. Results are expressed as PFU/mL. Each assay was performed in triplicate and the results are the means of the three replicates.The stability of vB_EaeM_φEap-3 at different pH levels was evaluated by suspending phages at approximately 1.2 × 10E. aerogenes strain 3-SP culture infected with vB_EaeM_φEap-3 was collected by low-speed centrifugation . Prior to DNA extraction, DNase (1 μg/mL) and RNase (1 μg/mL) were added to the phage lysate, which was then incubated at 37°C for 30 min. Following incubation, phage particles were precipitated with 1 M NaCl and 10% (w/v) polyethylene glycol (PEG) 8000 and incubated on ice for 1 h. The mixture was then centrifuged at 9000 ×g for 10 min at 4°C and the pellet was resuspended in 8 mL of SM buffer. An equal volume of chloroform was added to extract the PEG and cell debris, and then centrifuged at 4000 ×g for 15 min. The aqueous phase containing the bacteriophage particles was recovered and transferred to a new tube and DNA was extracted with phenol-chloroform  and precipitated with 100% ethanol. Finally, DNA samples were dissolved in 0.5 mL of sterile ddH2O and stored at 4°C.Cell debris from 500 mL of 1 using Basic Local Alignment Search Tool (BLAST) software  were identified using PHASTER3 .The genome of vB_EaeM_φEap-3 was sequenced using the Illumina HiSeq 2500 system . The reads were assembled using SSAKE (v3.8) assembly software. The final assembled sequences were searched against the protein and nucleotide databases available via the National Center for Biotechnology Information websitesoftware . BLASTP2PHASTER3 . The annSTER3  were used to evaluate the host range of vB_EaeM_φEap-3 (E. aerogenes strains (n = 18), with none of the other strains susceptible to infection. vB_EaeM_φEap-3 has a broader host range than the previous reported Enterobacter phage vB_EaeM_φEap-2 , followed by a rise period of 20 min. A growth plateau was reached within 40 min . The burResults obtained from temperature stability assays demonstrated that vB_EaeM_φEap-3 remained stable at temperatures ranging from 4–37°C. Decreases in infectivity were observed following incubation at 60 or 70°C for 15 min, while the phage was completely inactivated by incubation at 50°C for 60 min or 80°C for 15 min . Resultsn = 149, 53.6%) were present on the same strand. The shortest CDS encodes a putative protein of 26 amino acid residues (orf223), while the longest encodes a putative protein of 1394 residues (orf266). A specific function  could be assigned to 113 of the 278 predicted proteins (40.6%), with all sequences showing high identity to proteins from phages belonging to the “Kp15 virus” genus of the Tevenvirinae subfamily. No specific function was assigned to the remaining 165 CDSs , the deduced amino acid sequence of which shared 100% identity with the nucleoid disruption protein of Klebsiella phage KP15, suggested a lytic lifestyle for vB_EaeM_φEap-3 , with one transmembrane domain, was identified as a class III holin, and belongs to the holin T superfamily group  were detected . A tRNA s 59.4%; . No sequM_φEap-3 . ComparaM_φEap-3  and RB43M_φEap-3 , Klebsieo speeds , were alo speeds . The vB_o speeds . As a meo speeds . vB_EaeMly group . The lysly group .Myoviridae (A phylogenetic tree based on the predicted large terminase subunit amino acid sequences revealed that vB_EaeM_φEap-3 belongs to the genus “Kp15 virus” of the subfamily Tevenvirinae, family oviridae . This cloviridae .E. aerogenes, has become a major clinical challenge. As an important opportunistic pathogen, E. aerogenes can cause nosocomial outbreaks and invasive infections such as septicemia (E. aerogenes. Morphological characterization performed by TEM showed that vB_EaeM_φEap-3 is a member of the family Myoviridae, while phylogenetic analysis using previously verified markers (E. aerogenes.The spread of antibiotic-resistance among bacterial pathogens poses a serious problem in a clinical setting because of the lack of available treatment options. In particular, carbapenem resistance and its growing association with a multidrug-resistant phenotype in Enterobacteriaceae, including pticemia . In this markers  suggesteKT321315.The complete genome sequence of phage vB_EaeM_φEap-3 is available in GenBank under accession number JZ and ZZ did the experiments and contributed equally to this study as joint first authors. CT, XC, LH, XW, HL, WL, and AJ analyzed the data. RF, ZY, and JY provided the bacterial strains. XZ managed the project and designed the experiments. JZ and XZ wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cell viability was determined by CCK-8 assay, and the levels of autophagy and apoptosis were evaluated by Western blotting and caspase 3, 8, 9 activity assay. By administration with different concentrations of TGF-β1 , the effects of TGF-β1 on autophagy and apoptosis induced by H2O2 were observed, and the possible signaling pathways were also investigated by using various apoptosis inhibitors or an autophagy inhibitor Bafilomycin A (Baf A) in AF cells.AF cells were isolated from rat lumbar discs and subjected to different concentrations of exogenous H2O2 significantly impaired cell viability in a dose- and time-dependent manner. H2O2 also induced a sudden and the highest level of autophagy at 1 h, and gradually increased apoptosis through ERK pathway. The mitochondrial pathway was involved in H2O2-induced apoptosis in AF cells. TGF-β1 reduced the expression of p-ERK and downregulated the expressions of Beclin-1, LC3 II/I, and mitochondrial-related apoptotic proteins . Meanwhile, TGF-β1 downregulated the level of intracellular H2O2 through upregulating the expression level of glutathione peroxidase-1 (GPx-1).H2O2 through downregulating the expression of ERK in AF cells. TGF-β1 could downregulate the level of ERK and intracellular H2O2 by upregulating GPx-1.TGF-β1 reduced autophagy and apoptosis induced by exogenous H Chronic lower back pain (LBP) is an extremely common musculoskeletal disorder that affects approximately 80% of the population during their life . LBP is Although the etiology of LBP remains unclear, intervertebral disc degeneration (IVDD) is suggested as a main contributor. Annulus fibrosus (AF) is an important component of the intervertebral discs. During the degeneration of the intervertebral discs, the AF has been proven to be gradually lost due to apoptosis that consequently leads to its inability for withstanding to loading and induces the annular tears, fissures, and subsequent disc protrusions/herniations, ultimately resulting in the development of LBP –5. Thus,2O2) and hydroxyl radicals, nitric oxide (NO), etc. in degenerated intervertebral discs [Accumulating evidence has reported that there was excessive generation of reactive oxygen species (ROS), including superoxide anion, hydrogen peroxide  is a well-known factor to promote the cell growth and thus may be antagonistic to apoptosis of AF cells and alleviate the development of IVDD. This hypothesis has been preliminarily demonstrated by previous studies. Matsunaga et al. found that the expression of TGF-β1 was gradually decreased in AF cells of aging-induced IVDD model and almost disappeared in the 50-week-old mice . The add3) and firstly digested with 0.4% pronase  for 90 min at 37 °C, followed by second-digestion with 0.025% collagenase type II (Sigma) and 0.01% hyaluronidase type V (Sigma) overnight. The suspension was filtered through a 70-μm nylon mesh filter to remove the tissue debris. Then, AF cells were seeded into culture plates and maintained at 37 °C under a humidified atmosphere of 95% air and 5% CO2. The medium was changed every 2 days. First-passage cells in a monolayer were used throughout the experiments.All experimental protocols were approved by the Animal Care and Use Committee of Shanghai Jiaotong University and were in accordance with the guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health. After male, 6-week-old Sprague-Dawley rats were sacrificed by intraperitoneal administration of 10% chloral hydrate , the AF tissues were immediately excised from L1 to L6 intervertebral discs under aseptic condition. Primary AF cell cultures were prepared as our previously described , 17. Bri3 cells and maintained in complete medium . When the AF cells reached to 90% confluence, the medium was replaced with complete medium consisting of different concentrations of H2O2  for various time points . Each different concentration was repeated in 5 wells. To observe the influence of TGF-β1 addition on AF cells, different concentrations of TGF-β1  were added when the above pre-designed concentrations of H2O2 were reached. The cells were pretreated by various chemical blockers, including EK1/2 inhibitor U0126 , p38 inhibitor SB203580 , JNK inhibitor SP600125 , autophagy inhibitor Bafilomycin A , apoptosis inhibitors Z-VAD-FMK , Z-ATAD-FMK , and NS3694 , at 2 h before the addition of H2O2 and/or TGF-β1.AF cells were seeded into 96-well culture plates at a density of 5 × 102 incubator at 37 °C. Automicroplate reader  was used to measure the absorbance at a wavelength of 450 nm. Cell viability was expressed as the percentage of viable cells relative to untreated cells.Cell viability was measured using a Cell Counting Kit . In brief, 10 μL of CCK-8 solution was added to each well and incubated for 2 h in a humidified COWhen 90% confluence was reached, the AF cells were lysed in radio-immunoprecipitation assay buffer. The protein concentration of the whole cell lysate was measured by bicinchoninic acid protein assay kit . A total of 40 μg total protein from different samples was electrophoresed in different concentrations  of sodium dodecyl sulfate polyacrylamide gel and electro-transferred to a polyvinylidene difluoride membrane . After being washed for three times with 10 mM Tris-buffered saline with 1.0% Tween-20, the polyvinylidene difluoride membranes were incubated with 5% dehydrated skim milk to block nonspecific protein binding and then incubated with primary antibodies against PARP , cleaved caspase-3 , Bax , Bcl-2 , Beclin-1 , LC3 II/I , SQSTM1/p62 , T-P44/42 MAPK-ERK1/2 , SAPK/JNK , p38 , phospho-P44/42 MAPK-ERK1/2 , phospho-SAPK/JNK , phospho-P38 , cytochome C , caspase-9 , caspase-12 , GPx-1 , and β-actin  in the diluent  at 4 °C overnight, respectively. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies . Protein bands were visualized by the Super Signal Chemiluminescent Substrate system (Millipore or BeyoECL Plus) and quantified by densitometry analysis using the Bio-Rad Image Lab 2.0 software .405 (measured in each process hole)/A405 (measured in control hole).The activities of caspase-3, -8, and -9 were detected by using colorimetric activity assay kits , respectively. Cells were incubated in a lysis buffer containing 1% Dithiothreitol (DTT) on ice for 15 min and then centrifuged at 20,000 rpm for 15 min to isolate proteins. The protein concentration was determined using Bradford protein assay. A total of 200 μg protein lysate was incubated with 5 μL of caspase-3, -8, or -9 substrate and 1% DTT in the dark for 4 h at 37 °C. The activities of caspase-3, -8, and -9 were evaluated using a spectrophotometer at 405 nm with a microplate spectrophotometer . According to their concentration curve, the value was expressed by AThe AF cells harvested were re-suspended in a mixture of 500 μL culture medium and 500 μL fluorescent dye 5,5′6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1) , and then incubated in the dark at 37 °C for 20 min. Subsequently, the re-suspended cells were subjected to flow cytometry to determine the MMP (mtΔψ) which was measured by the dual-emission potential-sensitive probe. Cells with normal mitochondria had a high mtΔψ, JC-1 formed orange-red fluorescent J-aggregates; while in cells with depolarized or damaged mitochondria, the sensor dye appeared as green fluorescent monomers. The value of mtΔψ from each sample was expressed as the ratio of red fluorescence intensity/green fluorescence intensity.6 cells/mL. Following that, 100 μL of the solution (1 × 105 cells) was transferred to a 5 mL tube which was also added with 5 μL of Annexin V-FITC and 5 μL of PI. The cells were gently vortexed and incubated for 15 min at room temperature in the dark. Then, another 400 μL of 1× binding buffer was added into each tube, and flow cytometry analysis was performed within 30 min. The apoptosis incidence was calculated by the percentage of early apoptotic (Annexin V+/PI) cells plus the percentage of late apoptotic (Annexin V+/PI+) cells.Annexin V-FITC/PI apoptosis detection kit  was used to detect the apoptotic cells. The cells were collected by centrifugation  and re-suspended in 1× binding buffer at a concentration of 1 × 105 on 24-well plates under complete medium cultivation. After 60–70% confluence was reached, cells were transfected with 2 mg/mL GFP-LC3  or GFP-vector plasmid DNA using Lipofectamine 2000 . After incubation in Opti-MEM medium for 6 h, the cells were incubated in complete culture medium again for 24 h. The cells were fixed with 4% paraformaldehyde, and washed twice in cold PBS. Autophagy was evaluated by analyzing the formation of green fluorescent puncta of autophagosomes in GFP-LC3 transfected cells under the fluorescent microscope.AF cells were incubated at a density of 2 × 102O2  according to the manufacturer’s instructions.The total proteins or supernatants extracted from different examples were subjected to assay of catalase  and glutathione peroxidase (GPx-1)  activities as well as intracellular levels of Ht test in variables normally distributed, while Kruskal-Wallis and Mann-Whitney U test were adopted for variables with non-normal distribution. A P value < 0.05 was considered to be statistically significant.All data were expressed as mean ± standard deviation after at least three separate experiments and analyzed with SPSS13 Statistical Software . Differences between groups were analyzed using one-way ANOVA and independent Student’s 2O2 at different time points, the activity of AF cells was found to be significantly decreased, indicating its cytotoxicity. More importantly, the cell viability of AF cells were shown to be gradually declined with the increasing dosage of the H2O2  and the prolongation of stimulation time . After incubation with 50 μmol/L H2O2 for 4 h, the cells that survived still could reach 79%; but nearly half of the AF cells died using 100 μmol/L H2O2 (55%); toxic effect of H2O2 on AF cells was more obvious following treatment with 200 μmol/L H2O2 (43%). These findings indicated the toxic effects of H2O2 exhibited time- and dose-dependent patterns  were demonstrated to partially reverse the cytotoxic effects of H2O2 and improve the cell viability of AF cells to 59%, 67%, 76% at 4 h, respectively , thereafter gradually declined at 2 and 4 h . When the autophagic level reached peak (expressions of Beclin-1 and LC3II/I were highest), the level of p62 trimmed down to the minimum (P < 0.05); thereafter, p62 gradually increased with the decrease of autophagy  and 100 μmol/L H2O2 were used to incubate with the AF cells for 1 h. Western blot analysis showed that 10 and 20 ng/mL TGF-β1 could reduce the expressions of autophagy-related protein Beclin-1 and LC3 II/I, which was induced by H2O2, by a dose-dependent manner (P < 0.01)   after AF cells were stimulated by 100 μmol/L H2O2 for 1 h and the highest level was achieved at 4 h (P < 0.01) . Caspase-8 was slightly elevated (112%), but no statistical significance was observed     in the AF cells in vitro.As we know, there are many intracellular signaling pathways that can activate caspase-3 and excite downstream apoptotic processes in different cells: (1) oxidative stress could activate caspase-9, which belongs to mitochondrial pathway, and in turn activates the caspase-3 to execute the final apoptotic process ; (2) end2O2 incubation for 1 h were further examined. Western blot analysis showed that the expressions of phosphorylated ERK1/2, JNK, and p38 were significantly increased (P < 0.01) , caspase-3 blocker (Z-VAD-FMK), the mitochondrial pathway inhibitor (NS3694), and caspase-12 blocker (Z-ATAD-FMK). The Annexin V/PI double-staining experiments showed that U0126, Z-VAD-FMK, and NS3694 were able to significantly reduce H2O2-induced apoptosis in AF cells (P < 0.05) .Oxidative stress also activates a variety of mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, and p38. Regulation of protein kinases is often located at upstream of autophagic and apoptotic signaling pathways. Thus, the expressions of ERK1/2, JNK, and p38 under 100 μmol/L H01) Fig. a. The retor NS369, and castor NS369, and cas2O2 and exhibited a significantly statistical significance (P < 0.01)     on mitochondrial apoptosis pathway were further determined. Western blot analysis showed that TGF-β1 could effectively reverse Bax/Bcl-2 level raised by H2O2 (P < 0.05). Moreover, the anti-apoptotic effect was more significant when combining them (P < 0.01) than either of them (P < 0.05)   or ERK1/2 inhibitor U0126 (10 μM) alone or in combination with TGF-β1 (20 ng/mL) were used to incubate the AF cells for 1 h. Flow cytometry showed that TGF-β1 and Baf A could partially reverse the apoptosis induced by H2O2-induced cytotoxicity. Thus, the levels of intracellular H2O2 as well as GPx-1 and catalase activities were detected. It was found that, after exogenous H2O2 stimulation for 1 h, intracellular level of H2O2 was upregulated about 138% in AF cells than that in the control group    Fig. a. GPx-1 ly) Fig. b. The prly) Fig. c. Howeve2O2 was added to the AF cells to induce autophagy and apoptosis, and explore the protective roles of TGF-β1 on H2O2-treated cells. In line with our serum deprivation study [2O2 on the viability of AF cells by inhibiting autophagy (showing reduced GFP-LC3 autophagosomes accompanied with decreased expressions of Beclin-1 and LC3 II/I and increased p62) and apoptosis  at the early stage (0.5–4 h), preliminarily revealing that the supplementation of TGF-β1 may be an underlying treatment approach for IVDD [A lot of studies have demonstrated that the ROS, which is generated from dysfunctional mitochondria of disc cells, is an important reason for activating autophagy and then apoptosis, promoting disc degeneration –24. Hereon study , the prefor IVDD .2O2 in our study. The results indicated that the expressions of phosphorylated ERK1/2, JNK, and p38 were significantly increased after H2O2 treatment. However, only the ERK1/2 inhibitor U0126, not JNK and p38 blockers reversed the H2O2 apoptosis in AF cells, indicating that activation of ERK1/2 may be the main mechanism for H2O2-induced apoptosis of AF cells. Also, the use of TGF-β1 can achieve the same effects as U0126 and there was a synergistic effect between TGF-β1 and U0126 to suppress the ERK1/2, apoptosis, and autophagy, further illustrating that TGF-β1 may inhibit H2O2-induced apoptosis and autophagy by the ERK1/2 pathway. This seemed to be in line with previous studies in renal epithelial cells where apoptosis decreased if ERK was blocked [2O2 or TGF-β1 treatment is indispensable. In this study, we found that the apoptosis incidence was significantly declined after the use of autophagy inhibitor Baf A and the effect was more obvious when the combination of Baf A and TGF-β1, implying that the increased autophagy may promote apoptosis and TGF-β1 may inhibit apoptosis by blocking autophagy. This finding seemed to be in accordance with our previous study in nucleus pulposus cells [Oxidative stress can activate the mitogen-activated protein kinases (MAPKs) pathway , includi blocked , 32, but blocked . This ma blocked . Moreove blocked . Some st blocked , 36, but blocked , 38. Thu2O2 treatment may be attributed to the lower generation of antioxidants and TGF-β may alleviate ROS-induced injuries in AF cells by increasing the formation of antioxidants. In this study, the activity of GPx-1 was also detected. As expected, the GPx-1 activity and expression were significantly enhanced after the addition of medium-high dosed TGF-β1, which was also coincident with the previous studies in colon cancer cells [The previous studies reported that ROS generated within cells can be neutralized by superoxide dismutase, glutathione peroxidase, catalase or other non-enzymatic antioxidants and glutathione, etc., and achieved the balance of oxidation-reduction in cells , 40. Thuer cells . The reger cells , 43, alt2O2 had significant cytotoxicity, and promoted autophagy and apoptosis through ERK pathway in rat AF cells. TGF-β1 could reduce the toxic effects of H2O2 to the AF cells through inhibiting ERK pathway to inhibit excessive autophagy and then apoptosis induced by H2O2. The protective effects of TGF-β1 on rat AF cells may also be achieved by upregulating GPx-1 and reducing the level of intracellular H2O2. These findings indicated the supplementation of TGF-β1 (or drugs that induced the increase of TGF-β1) may be an underlying approach for prevention and alleviation of AF cell loss and subsequent development and progression of IVDD and LBP in clinic.Our findings suggested that exogenous H


Pueraria plants, has various medical functions. Cytochrome P450s (CYPs) are crucial phase I metabolizing enzymes, which have been spotlighted for their effects on drug metabolism. The interaction between puerarin and CYPs (CYP1A2 and CYP2D6) was investigated by fluorescence, UV-Vis and circular dichroism spectroscopies, as well as molecular docking, to explore the underlying mechanism under simulated physiological conditions. The molecular docking results indicated that puerarin interacted with CYPs mainly by hydrophobic force and hydrogen bonding. The fluorescences of CYPs were quenched statically. Binding constants (Ka) and number of binding sites (n) at different temperatures were calculated, with the results being consistent with those of molecular docking. At the same temperature, puerarin bound to CYP1A2 more weakly than it did to CYP2D6. UV-Vis and circular dichroism spectroscopies confirmed the micro-environmental and conformational changes of CYP1A2 and CYP2D6. The findings provide reliable evidence for clarifying the structures and functions of CYPs.Puerarin, an isoflavone glycoside extracted from  Pueraria plants, can lower blood pressure, resist oxidation, arrhythmia and inflammation, as well as inhibit tumor cell proliferation  is the concentration of the quencher. Plotting F0/F vs. [Q] indicates the types of quenching, i.e., a straight line for individual static or dynamic quenching, and an upward curve for mixed types  vs. lg[Q] at different temperatures [The apparent binding constant Keratures ,14,15  and entropy change (ΔS) during binding can be considered constants at an almost invariable temperature, which can be calculated by the van’t Hoff equation lnKa = −ΔH/RT + ΔS/R and binding equation ΔG = ΔH − TΔS. Thermodynamic parameters were calculated by the binding constants in Drugs commonly interact with biomacromolecules via hydrogen bonding, hydrophobic interactions, van der Waals force and electrostatic attraction, et al. classified the interaction forces between drugs and protein by ΔH and ΔS into hydrophobic interaction , van der Waals force  and electrostatic attraction  [Ross  ΔS > 0) .Since the ΔG values of the two systems were lower than zero, puerarin interacted with CYPs following thermodynamically spontaneous reactions. Moreover, the positive results of ΔH and ΔS indicate puerarin bound to the two CYPs by hydrophobic forces. Puerarin, as a small hydrophobic molecule, intercalated the hydrophobic regions inside CYPs. In addition, hydrogen bonding cannot be ruled out due to the multiple hydroxyl groups of puerarin.Proteins exhibit UV absorptions owing to Trp (280 nm), Tyr (280 nm) and Phe (257 nm) as well as peptide bonds (225 nm). After adding drugs, the absorptions of protein chromophores are bound to change . As exhiCD spectra were determined to observe the interactions of puerarin with CYPs and corresponding structural changes . CYPs ha−1, were selected. Cluster analysis disclosed that the most probable conformation had the energy of −7.3 kcal·mol−1, and 50% docked conformations were included, inferring that the docked structure was reliable and stable. The docking results were also visualized by Ligplus and Autodock  encapsulating the protein active center, a hydrophobic cavity, was constructed, mainly including F226, I314, G316 and A317 residues on α−helix as well as F451 and G459 residues on random coils. Puerarin entered the active center by hydrophobic interactions and hydrogen-bound to R456, I459 and W133.−1. Meanwhile, the binding affinity was predicted as ~μM. The results confirm that the docked structure was reliable and stable. The docking results were also visualized by Ligplus and Autodock , UV-Vis spectrometer , CD spectrometer  and pH meter .−6 mol·L−1, BD, stored at −80 °C, thawed at room temperature immediately before use), methanol , sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride . Double-distilled water was used throughout the experiments.The reagents included puerarin , CYP1A2, CYP2D6 (1 × 10−3 mol·L−1 puerarin solution: Puerarin (0.0104 g) was placed in a 25 mL volumetric flask, diluted with HPLC-grade methanol and stored at 4 °C.(1) Preparation of 1×10−1 PBS buffer: NaH2PO4 (0.5928 g) and Na2HPO4 (5.8019 g) were placed in a 100 mL volumetric flask and diluted with double-distilled water. The solution (10 mL) was then transferred to another 100 mL volumetric flask, diluted with double-distilled water, pH-adjusted to 7.4, and added 0.5844 g NaCl (0.1 mol·L−1) to maintain ionic strength.(2) Preparation of 0.02 mol·L−3 mol·L−1 puerarin solution to the final concentrations of 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 × 10−5 mol·L−1. Being excited at 280 nm, fluorescence spectra were recorded from 290 to 440 nm at 293 K and 303 K respectively.PBS (2 mL) and 10 µL of CYP1A2 (or 3 µL of CYP2D6) were added in a quartz cuvette, to which were added 10 µL aliquots of 1 × 10−3 mol·L−1 puerarin solution. UV-vis spectra were measured from 220 to 400 nm.PBS (2 mL) and 10 µL of CYP1A2 (or 10 µL of CYP2D6) were added in a cuvette, to which were added 10 µL aliquots of 1 × 10−4 and 9 × 10−4 mol·L−1. By using the corresponding blank solutions as the backgrounds, CD spectra were measured from 190 to 280 nm. Conformations were calculated using the built-in JASCO-810 software.Various amounts of puerarin solution and 10 µL of CYP1A2 (or 10 µL of CYP2D6) were added in a quartz cell and diluted with PBS buffer to 1000 µL to the final puerarin concentrations of 0, 8 × 10The binding sites and forces between puerarin and CYPs were predicted by molecular docking. The crystal structures of protein receptors human CYP1A2 (entry: 2HI4) and human CYP2D6 (entry: 3TBG) were obtained from the Protein Data Bank. The structure of puerarin was drawn by ChemOffice 11.0, optimized to the structure with lowest energy, and then processed by Autodock through adding hydrogens, calculating electric charges and docking with the protein receptors.In summary, the interactions between puerarin and two CYPs were studied by fluorescence, UV-Vis and circular dichroism spectroscopies together with molecular docking. Puerarin quenched the fluorescences of CYP1A2 and CYP2D6 statically, and bound to them mainly by hydrophobic force and hydrogen bonding. At the same temperature, puerarin bound to CYP1A2 more weakly than it did to CYP2D6. Furthermore, puerarin altered the conformations of CYP1A2 and CYP2D6. The findings provide reliable evidence for investigating the interactions between compounds and CYPs as well as for clarifying the structures and functions of CYPs.


Xylan, the major hemicellulosic component, contributes to the structural stability of wood and represents a tremendous quantity of biobased chemicals for fuel production. Monomeric xylose conversion to value-added chemicals such as furfural, xylitol, and xylonic acid could greatly improve the economics of pulp-paper industry and biorefinery. Acetic acid (HAc) is used as a friendly and recyclable selective catalyst amenable to xylan degradation and xylooligosaccharides production from lignocellulosic materials. However, HAc catalyst usually works much feebly at inert woods than agricultural straws. In this study, effects of different iron species in HAc media on poplar xylan degradation were systematically compared, and a preferable Fe3+, xylose yield ranged between 72.5 and 73.9%. Additionally, pretreatment was effective in poplar delignification, with a lignin yield falling between 38.6 and 42.5%. Under similar conditions, saccharification efficiency varied between 60.3 and 65.9%. Starting with 100 g poplar biomass, a total amount of 12.7–12.8 g of xylose and 18.8–22.8 g of glucose were harvested from liquid streams during the whole process of Fe3+-HAc hydrolysis coupled with enzymatic saccharification. Furthermore, the enhancement mechanism of Fe3+ coupled with HAc was investigated after proof-of-concept experiments. Beechwood xylan and xylose were treated under the same condition as poplar sawdust fractionation, giving understanding of the effect of catalysts on the hydrolysis pathway from wood xylan to xylose and furfural by Fe3+-HAc.In presence of 6.5% HAc with 0.17–0.25 wt% Fe3+-assisted HAc hydrolysis process was demonstrated as an effective approach to the wood xylose and other monosaccharides production. Synergistic effect of Lewis acid site and aqueous acetic acid provided a promising strategy for catalytic valorization of poplar biomass.The Fe Plant biomass has been suggested as an abundant and sustainable source for the future chemicals, fuels, and materials due to its carbon-neutral nature and renewability , 2. BiomO-acetyl substitutions and 4-O-methyl α-d-glucosyluronic acid in the hemicelluloses fractions making it susceptible to the thermal hydrolysis  Cl/HCl + FeCl3 (4.8 wt%) + acetone–water system and subsequent saccharification, which suggests that enough water washing on pretreated biomass would eliminate the detrimental effects of soluble catalysts and lignocellulose-derived inhibitors on microbial growth. Additionally, it is expected to recover ferric chloride by adjusting pH with sodium hydroxide until the formation of ferric hydroxide precipitation. Then vacuum filtration could be used to separate and recover ferric hydroxide solid. Furthermore, the recovered ferric chloride can participate in Fenton reaction for the disposal of wastewater  increased from 0.02 to 0.08 wt%, indicating that high concentrations of FeCl3 caused more xylan conversion. FeCl3 exerted different effects on XOS, xylose, and furfural production during the hydrolysis process. For instance, it was observed that XOS yields declined from 31.0 to 14.4%, while the generated xylose and furfural increased from 24.2% and 3.4% to 52.3% and 8.3%, respectively, after being treated by the elevated concentration of Fe3+ salt. These results suggest that Fe3+ could promote the decomposition of xylan and further degrade oligosaccharides into monosaccharides and other by-products, such as furfural  ranging from 0.02 to 1.32 wt% on poplar xylan degradation was also investigated with a concentration of 6.5% HAc. As illustrated in Fig. 3+ as an enhancer of HAc on xylose production, generating by-products XOS and furfural during xylan conversion process were rigorously examined. It was observed that xylose yield increased from 25.0 to 73.9% as [Fe3+] ratio increased from 0.02 to 0.25 wt%, but a further increase of [Fe3+] to 1.32 wt% decreased the yield (8.8%). Similarly, the potential xylan showed an increasing trend, then decreasing with the maximum value of 91.1% at 0.25 wt% of [Fe3+], indicating faster rate of xylose degradation than the production rate of furfural. Meanwhile, when [Fe3+] reached 0.25 wt%, XOS decreased to zero, indicating that [Fe3+] could accelerate the decomposition of dissolved oligomeric xylose into monomeric xylose [3+] ratio favored the xylose formation, but an excessive Fe3+ resulted in more degradation products derived from monomeric xylose. Additionally, furfural yield dropped from 32.5 to 29.3% as [Fe3+] increased from 0.66 to 1.32 wt%. Liu and Wyman , HAc media enabled XHR generate maximum xylose yields of 72.5% and 73.9%, respectively.The influence of  prior to and immediately after the pretreatment were also recorded. The pH value corresponding to each added Fe3+ concentration is displayed in Fig. ∆ = 1.4 pH units) occurred as [Fe3+] increased from 0.02 to 1.32 wt%, indicating the presence of Fe3+ could contribute an enhancement in the acid strength of the aqueous acetic acid solution. Notably, the highest xylose production can be obtained at the initial pH value around 1.3 corresponding to 0.25 wt% concentration of Fe3+ which has proven to be preferable for the effective pretreatment. Nonetheless, following hydrolysis, prehydrolysis pH values increased compared to initial pH prior to pretreatment. For instance, as presented in Fig. 3+] into the reaction media. In general, a low pH will drive the pretreatment and the ash buffering capacity will mitigate the reduction in pH that results from addition of acid. Notably, some minerals such as KCl, K2HPO4, and K2SO4 in biomass could realize the pH-mediated abirritation ) are acidic (pH 1.3). To assess whether or not the xylan conversion was dominated by the acidity of Brønsted acid, control experiments using acid to adjust pH in aqueous HAc with an identical proton concentration were implemented. Results show that the potential xylan (91.1%) with Fe3+-HAc was significantly higher than that with pH-adjusted HAc (61.6%) at the same acidity. Consistently, higher xylose (73.9%) in the case of Fe3+ was obtained compared with pH-adjusted solution (31.8%). This illustrates the activity of Fe3+ in xylan conversion  was not solely governed by its Brønsted acidity. In a separate study, Marcotullio and De Jong  (from 0.08 to 0.25 wt%), it was seen that the concentrations of xylose (from 20.1 to 23.5 g/L) and furfural (from 8.3 to 13.7 g/L) were increased. It was worthwhile to mention that the formation rate of xylose is lower at higher concentrations of Fe3+ (from 0.08 to 0.25 wt%), indicating the faster degradation of xylose to furfural.To understand the effect of catalysts on the hydrolysis pathway from wood xylan to xylose and furfural by Feon Table . For ins3+ Table , both xynd Mazza  found th3+  was achieved compared with HAc alone. Catalytic mechanisms were verified by employing model compounds of xylan and xylose. This study provides a deep understanding of the metal–organic hydrolytic conversion of wood polysaccharides to valuable products.Fe3·6H2O, FeSO4·7H2O, Fe3O4, sulfuric acid and xylan  used in the experiments were analytical reagents without further purification. Xylose, xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) were purchased from Shanghai Yikuo Instrument Co., Ltd. , and were used as standards.A fast-growing poplar tree (3 years old) was harvested from the Jiangsu province in China. The wood sample was ground to pass through a 20-mesh size screen (< 0.85 mm), air-dried, homogenized in a single lot, and stored at room temperature. The moisture content of milled sawdust was approximately 10% based on total wet biomass weight. The composition of the poplar sawdust is glucan (43.3%), xylan (17.6%), acid-insoluble lignin (3.7%), acid-soluble lignin (24.2%), and ash content (0.6%) by dry weight. HAc, FeClA series of experiments were carried out in a sealed 30 mL stainless steel tube reactor using diluted HAc as the catalyst in presence of different species of Fe ions. The biomass was thoroughly mixed with a certain amount of HAc and varying concentrations of Fe species in the digester and then pre-impregnated for 1 h at room temperature. Based on our previous optimization, the reaction temperature, time and HAc concentration were chosen to be 170 °C, 27 min and 6.5% (v/v), respectively . In each® 1.5 L, Novozymes Inc.). The enzyme loading was 20 FPU/g glucan at 50 °C and 150 rpm in an orbital shaker (New Brunswick) [Following pretreatment, enzymatic hydrolysis was carried out at 5% (w/v) for 96 h. The recovered solids were mixed with 50 mM sodium citrate buffer at pH 4.8, 0.2% (w/v) tetracycline, and the cellulose enzyme  . Samples2SO4 at a flow rate of 0.6 mL/min and column temperature of 55 °C. Xylooligosaccharides were determined using an HPAEC-PAD (Dionex ICS-5000) equipped with a carbopac PA-200 as the anion-exchange column and the separation method used herein was reported elsewhere [The chemical compositions of all the material were determined according to the standard laboratory analytical procedures developed by the National Renewable Energy Laboratory . The moilsewhere . BrieflyThe xylose, XOS, furfural yield, enzymatic efficiency, remaining glucan and xylan, xylan loss and weight loss were calculated according to the following equations.


Supplemental Digital Content is available in the text Previous investigations yielded inconsistent results for the associations between pancreatic cancer (PC) risk and genetic polymorphisms. The study aimed to perform a systematic review and meta-analysis of studies exploring association of some genetic polymorphisms and PC risk.We systematically searched on PubMed and Web of Science for association of genetic polymorphisms and PC risk published from 1969 to January 2019. We computed the multivariate odd ratio (OR) and 95% confidence intervals (CI), comparing different genetic types.Arg399GIn and Arg194Trp, excision repair cross complementation 1 (ERCC1) rs11615 and rs3212986, ERCC2 rs13181) polymorphisms and PC risk.The present meta-analysis showed significant associations between deoxyribonucleic acid (DNA) repair gene (X-ray repair cross-complementing group 1 (XRCC1) Because of the limited sample size and ethnicity enrolled in the present meta-analysis, further larger scaled studies should be performed to demonstrate the association. But there is still no standard program for screening patients at high risk of PC. It is known that the development of PC is a complex and multifactorial process. Many factors, such as smoking, drinking, diabetes, obesity, body mass index, as well as environmental chemicals, are known to play a key role in PC development.,5 Nevertheless, hereditary factors could not be ignored and might play an essential role in PC development. Emerging evidence suggests that the human genes for metabolism (cytochrome P-450 1A1 (CYP1A1), glutathione S-transferase M1 (GSTM1), glutathione S-transferase T1 (GSTT1), N-acetyltransferase 2 (NAT2), UDP glucuronosyltransferase (UGT1A7), methylation gene (Methylenetetrahydrofolate reductase (MTHFR)), inflammatory response gene (tumour necrosis factor (TNF)-α), deoxyribonucleic acid (DNA) repair gene (X-ray repair cross-complementing group 1 gene (XRCC1), 8-oxoguanineDNA glycosylase (OGG)1, excision repair cross complementation (ERCC) 1, ERCC2), alcohol-metabolizing enzyme gene  2), and another gene ) are most important candidate genes for influencing the risk of PC, and genetic polymorphisms in these gene might be associated with PC risk. However, previous investigations yielded inconsistent results for association of these genetic polymorphisms and PC risk. In the present investigation, we aim to perform a systematic review and meta-analysis to evaluate the association of these genetic polymorphisms and PC risk. In addition, we conducted subgroup studies according to different ethnicities.Pancreatic cancer (PC) is one of the most fatal malignant tumors.2. We supplied a PRISMA 2009 checklist. Ethical approval was not applicable in the study.This study was conducted on the basis of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement 2.1Articles on genetic polymorphism and PC risk were searched for in PubMed and Web of Science databases until January 2019. Search terms were the following: (“pancreatic cancer” OR “pancreatic carcinoma” OR “pancreatic neoplasm”) AND (“gene” OR “polymorphisms”). After that, duplicates were removed. A total of 71 articles were screened in our study.2.2Our study included all articles exploring an association between gene polymorphisms and PC risk in adult humans. The study included in the study should be a case-control or cohort study. Additionally, studies should include both controls and PC patients as participants. All included studies reported odds ratio (OR) or data from which OR could be calculated. Moreover, there were no restrictions on language.Articles were eliminated while they were associated endocrine neoplasms of the pancreas, familial PC, and hereditary PC syndrome. Secondary processing of literature such as reviews and meta-analysis articles were dropped. Case studies without group-level statistics were excluded.2.3Titles and abstracts of articles were read by 2 different individuals. According to the inclusion and exclusion criteria, 71 articles were selected to read full-texts. We recorded following data from these full-texts: Author, publication years, country of origin, ethnicity, gene polymorphisms studied, mean age (standard deviation (SD)) of cases and controls, numbers of case and control populations, pathology of diseases, the exact genotyping techniques, genotyping quality control measures, evidence of the Hardy–Weinberg equilibrium (HWE), and variables for which statistical adjustments were made.2.4Q test, and evaluated the amount of variation derived from heterogeneity with computed I2. We conducted fixed effects models to generate summary effect size in absence of heterogeneity  of included studies. Inversely, with invariably high heterogeneity between studies, we performed random effects models to summarize effect size. Subgroup analysis for different ethnicities was conducted to observe the effect of heterogeneities for ethnicities to the heterogeneity of meta-analysis. All statistical analysis was conducted with STATA 12.0 software.We conducted meta-analysis to summarize results while at least 3 articles were presented for the strength of association between each polymorphism and PC risk. We computed the multivariate ORs and 95% CI. We assessed heterogeneity between studies with 33.1http://links.lww.com/MD/D142 showed the study characteristics and results of the included 71 studies. We collected data from 6 XRCC1 (n = 1240 cases and 3918 controls),,17–21 5 OGG1 (n = 1714 cases and 3683 controls),,17,22–24 4 MTHFR (n = 957 cases and 1766 controls),,25–27 4 ERCC1 (n = 934 cases and 1039 controls),28–30 and 6 ERCC2 (n = 1759 cases and 2050 controls),23,28–31 studies.Figure 3.2I2 = 0.0%, P = .733; AA vs GG: OR = 1.58, 95%CI 1.22–2.06, I2 = 0.0%, P = .595; GA+AA vs GG: OR = 1.38, 95%CI 1.15–1.64, I2 = 1.7%, P = .383. Arg194Trp: CT vs CC: OR = 1.22, 95%CI 1.04–1.44, I2 = 2.3%, P = .402; TT vs CC: OR = 1.25, 95%CI 1.08–1.43, I2 = 0.0%, P = .754; CT+TT vs CC: OR = 1.22, 95%CI 1.02–1.46, I2 = 45.7%, P = .137) .Six of the studies investigated XRCC1 Arg399GIn and XRCC1 Arg194Trp polymorphisms using 1240 cases and 3918 controls. Figure I2 = 0.0%, P = .698; CC vs GG+GC: OR = 0.93, 95%CI 0.75–1.11, I2 = 0.0%, P = .821; CC+GC vs GG: OR = 0.99, 95%CI 0.86–1.11, I2 = 71.0%, P = .008) .Five of the studies explored OGG1 Ser326Cys polymorphism using 1714 cases and 3683 controls. Figure I2 = 0.0%, P = .763; TT vs CC: OR = 1.54, 95%CI 1.10–2.16, I2 = 5.8%,  = .364; ERCC1 rs3212986: GT vs GG: OR = 1.34, 95%CI 1.06–1.69, I2 = 0.0%,  = .953; TT vs GG: OR = 2.29, 95%CI 1.58–3.31, I2 = 0.0%,  = .982)  .Four studies investigated ERCC2 rs13181 polymorphism using 1759 cases and 2050 controls. Figure 3.3I2 = 68.6%, P = .023; TT vs CT: OR = 1.39, 95%CI 0.70–2.07, I2 = 67.9%, P = .025; TT+ CT vs CC: OR = 0.79, 95%CI 0.57–1.01, I2 = 30.6%, P = .229; TT vs CT+CC: OR = 0.86, 95%CI 0.41–1.32, I2 = 89.4%, P < .001; MTHFR A1298C: TT vs CC: OR = 0.84, 95%CI 0.46–1.21, I2 = 43.6%, P = .170; TT vs CT: OR = 0.79, 95%CI 0.44–1.14, I2 = 41.8%, P = .179; TT+ CT vs CC: OR = 0.80, 95%CI 0.41–1.19, I2 = 49.0%, P = .141; TT vs CT+CC: OR = 0.99, 95%CI 0.78–1.19, I2 = 0.0%, P = .448)  polymorphisms and PC risk. In addition, further subgroup analysis by ethnicity indicated that there was a statistically significant association between XRCC1 Arg399GIn and Arg194Trp, ERCC2 rs13181 polymorphisms and PC risk in Asians under all genetic models.,33 NER pathway could identify the site of damage, unwind the DNA duplex around the site, cut the DNA upstream and downstream of the damaged area, and repair the gap.,35 The influence of NER gene polymorphisms on PC risk is not quite well studied. In the present study, polymorphisms of 3 NER genes investigated  were demonstrated to be associated with PC risk. The XRCC1 is located on chromosome 19q13.2-13.3. XRCC1 shows a variety of single nucleotide polymorphisms, of which those in the tenth and sixth exons are common and lead to Arg399GIn and Arg194Trp amino acid substitutions, respectively. The present study showed a result distinct from a recent meta-analysis, which suggests that the XRCC1 Arg194Trp genetic polymorphism is not significantly associated with PC risk. In these included studies, Jiao et al indicated no significant associations between XRCC1 Arg194Trp polymorphism and PC risk. Wang et al reported no significant differences in PC risk in participants with different XRCC1 Arg194Trp genetic polymorphism. Nakao et al observed no significant associations between PC risk and XRCC1 Arg194Trp polymorphism. In addition, Hou et al showed no significant relation between XRCC1 Arg194Trp polymorphism and PC risk. Wang et al reported that Arg194Trp polymorphisms did not affect PC risk. However, Yan et al indicated that the XRCC1 Arg194Trp genetic polymorphism might be associated with the risk of PC.  The result of the present meta-analysis might be influenced by the article published by Yan et al. The present study included further larger scaled studies, which caused the inconsistent result. ERCC1 and ERCC2 are DNA repair genes with the chromosomal locus 19q13.3. They could reverse ionizing radiation-induced damage and DNA damage by chemotherapy.,39 A recent study indicated that ERCC2 rs13181 polymorphism might be important in stimulating the development of PC, especially for Asians. Our result was consistent with the study. He et al indicated no significant associations between ERCC2 rs13181 polymorphism and PC risk. Ying et al reported no significant associations between ERCC2 rs13181 polymorphism and PC risk.  However, Sileng et al indicated that ERCC2 rs13181 polymorphism exposed higher risk to PC. In addition, McWilliams et al indicated that ERCC2 rs13181 polymorphism was associated with PC risk.  Zhao et al showed that ERCC2 rs13181 gene polymorphisms contribute to the development of PC.  The inconsistent results from different articles could not be explained by the ethnicity, research type or numbers of participants. But the present study provided a chance to summary the results in these articles. In addition, the summarized result is consistent with other meta-analysis. The present study showed significant associations between DNA repair gene polymorphisms  and PC risk. On the basis of genetic profiles of PC, genomic instability mediated by DNA repair deficiency is a crucial event in development of PC. DNA repair machinery plays an important role in defending cells against environmental hazards like ultraviolet (UV) rays, ionizing radiation, diet, and smoking. A key DNA repair mechanism, nucleotide excision repair (NER), can have an impact on gene–gene rearrangement, deletion, translocation, and amplification.,42 These included studies showed inconsistent results, which might be caused by different folate status of ethnic differences and the environment in which they lived in. Result of the present study was corresponding to a recent study, which indicated that MTHFR polymorphisms (C667T and A1298C) are not associated with PC risk.The present study showed no significant association between MTHFR polymorphism and PC risk. MTHFR is a crucial enzyme within the folate methionine pathway. Folate intake increases plasma folate and reduces total homocysteine concentration, which may reduce and risk of cancer. In addition, the possible mechanism for the effect of MTHFR on PC is that DNA methylation, which might be associated with PC risk. Considering the limited sample size enrolled in this meta-analysis, further larger scaled studies are essential to provide a more precise estimation on the association in Caucasians. Subgroup study indicated no significant associations between OGG1 Ser326Cys polymorphism and PC risk in Caucasians. In addition, Nakao et al indicated no significant associations between OGG1 Ser326Cys polymorphism and PC risk in Asians. Subgroup study indicated significant associations between ERCC2 rs13181 polymorphism and PC risk in Asians, whereas the associations were not obvious between ERCC2 rs13181 polymorphism and PC risk in Caucasians. The differences between Asians and Caucasians may be partly result from the different genetic backgrounds and environments or lifestyles. The result is corresponding to a recent meta-analysis. This study showed significant associations between XRCC1 Arg399GIn and Arg194Trp genetic polymorphisms and PC risk in Asians, whereas Jiao et al indicated that no significant associations between XRCC1 Arg399GIn and Arg194Trp genetic polymorphisms and PC risks in Caucasians.There were some limitations in the study. Firstly, the present study only included case-control studies. No prospective cohort studies were included in the study. Second, only 7 studies had a population-based design in the selection of cases. Most of the studies were from hospitals.5In conclusion, the present meta-analysis suggested significant associations between DNA repair gene  polymorphisms and PC risk. Because of the limited sample size and ethnicity enrolled in the present meta-analysis, further larger scaled studies should be performed to demonstrate the association.Data curation: Peng Dai, Jing Li, Weibin Li, Xueliang Qin, Xiaoyong Wu, Weidong Di, Yanzhong Zhang.Methodology: Peng Dai, Xueliang Qin, Xiaoyong Wu, Weidong Di.Writing – original draft: Peng Dai.Writing – review & editing: Peng Dai.Investigation: Jing Li, Weibin Li.Software: Yanzhong Zhang.