Furthermore, a non-metabolizable isosteric analog of vitamin E which retains antioxidant activity neither inhibited 15-lipoxygenase nor prevented ferroptosis. These results call into question the prevailing model that vitamin E acts predominantly as a non-specific lipophilic antioxidant. We propose that, similar to the other lipophilic vitamins A, D and K, vitamin E is instead a pro-vitamin, with its quinone/hydroquinone metabolites responsible for its anti-ferroptotic cytoprotective activity.Ferroptosis is a form of programmed cell death associated with inflammation, neurodegeneration, and ischemia. Vitamin E  has been reported to prevent ferroptosis, but the mechanism by which this occurs is controversial. To elucidate the biochemical mechanism of vitamin E activity, we systematically investigated the effects of its major vitamers and metabolites on lipid oxidation and ferroptosis in a striatal cell model. We found that a specific endogenous metabolite of vitamin E, alpha-tocopherol hydroquinone, was a dramatically more potent inhibitor of ferroptosis than its parent compound, and inhibits 15-lipoxygenase via reduction of the enzyme’s non-heme iron from its active Fe We propose that the anti-ferroptotic effect of vitamin E results from a four-step biochemical mechanism: 1) oxidative hydrolysis of alpha-tocopherol to alpha-tocopherol quinone; 2) reduction of alpha-tocopherol quinone to alpha-tocopherol hydroquinone; 3) inhibition of 15-LO via reduction of its non-heme Fe3+ center to the inactive Fe2+ state by alpha-tocopherol hydroquinone; and 4) inhibition of the ferroptosis cascade by blocking formation of lipid peroxidation products. These results support a reinterpretation of the mechanism of vitamin E, which has previously been attributed to a non-specific antioxidant activity. As with the other fat-soluble vitamins , our data suggest that vitamin E is a pro-vitamin that is converted to an active form, vitamin E hydroquinone, which functions at a specific set of targets. These findings link vitamin E’s purported clinical effects to an anti-ferroptotic mechanism-of-action.Ferroptosis is a form of iron-dependent, lipid oxidation-mediated programmed cell death implicated in a wide array of disease pathologies including inflammation, ischemia-reperfusion injury, and neurodegeneration –5. Vitam3 and extracted 3x with ethyl acetate. The organic phase was dried over Na2SO4 and concentrated in vacuo. The residue was purified by silica gel chromatography to obtain pure product.To a round-bottomed flask, the chroman  was dissolved in a 2:1 acetonitrile:water mixture (15 mL). This solution was cooled to 0°C. A solution of ceric ammonium nitrate (2.1 eq) in water (5 mL) was added dropwise. The reaction mixture was stirred at 0°C for 2 h. Upon reaction completion, the mixture was neutralized with sat. aq. NaHCO2. Lindlar catalyst  or 10% Pd/C  (0.1 eq by weight) was added and the vessel was purged again with H2. The resulting suspension was stirred at room temperature overnight under a H2 atmosphere. Upon reaction completion, celite was added. The resulting slurry was filtered through a fritted funnel. The filter cake was washed successively with THF. The filtrate was concentrated in vacuo to obtain pure product.To a 20 mL septum-capped vial, the vitamin E quinone  was dissolved in tetrahydrofuran (THF) (15 mL). Then the vessel was purged with HHdhQ7/Q7; immortalized by SV large T antigen) were obtained from Coriell. Cells were cultured in high-glucose (25 mM) DMEM (Gibco) medium supplemented with fetal bovine serum , penicillin , and streptomycin . For routine maintenance, Q7 culture medium also contained Geneticin Sulfate . Cells were routinely passaged every 3–4 days, maintaining sub-confluent cell densities.Mouse striatal Q7 cells -RSL3 that most potently (IC50 ~200 nM) inhibited Q7 cell viability. To assess compound rescue activity and potency from ferroptotic cell death, Q7 cells were seeded in clear bottom, black wall tissue culture-treated polystyrene plates (Corning) using either an electronic multichannel pipette or a Multidrop™ Combi Reagent Dispenser (ThermoFisher Scientific). Cells were incubated for 5 hours at 33°C in an atmosphere with 95% humidity and 5% CO2 to allow attachment of the cells to the culture plate. Test compounds were administered to the desired final concentrations using a D300e Digital Dispenser (Tecan), followed within 15 minutes by RSL3 . DMSO diluent was back-filled to a final concentration of 0.3% (v/v). Cell plates were incubated at 33°C  for 18 hours. After a 15-minute equilibration to room temperature, cell number/viability was assessed using the CellTiter-Glo® 2.0 Cell Titer Glo 2.0 reagent  which was added using the Multidrop™ Combi Reagent Dispenser (Thermo Fisher Scientific). After 15 minutes of incubation at room temperature, the luminescence (100 ms integration time) per well was determined using the BioTek Synergy plate reader. Data was imported into and analyzed using Dotmatics Studies software suite. EC50 values for each compound were estimated using standard four-parameter curve fitting algorithms. pEC50 is defined as negative-log10 (EC50) in M units. RSL3 treatment typically reduced cell number/viability by >80%. The assay performance was assessed by Z-prime calculations on each assay plate, with observed Z-prime values of >0.7. We also note that maximally efficacious concentrations of the vitamin E compounds restored RSL3-treated cell viability to levels comparable to cells that had not been treated with RSL3, indicating complete rescue of cell viability by these compounds.Ferroptotic cell death was induced in Q7 cells using RSL3, a GPX4 inhibitor previously described . All fouFerroptosis was induced by RSL3  in Q7 cells as described above. The protective activity of co-treated compounds was assessed also as described above. The rate of cellular lipid oxidation after GPX4 inhibition was assessed by monitoring the time-dependent changes in green fluorescence of cells pre-labeled with BODIPY™ 581/591 C11 dye  using the IncuCyte S3 live-cell imaging apparatus (Sartorius) using a 10x objective. Cells  were imaged once hourly for up to 18 hours after compound and RSL3 addition. Quantification of cell images was performed using the IncuCyte Zoom software using the Basic Analyzer tool which utilized an algorithm that calculated the total green-positive area per well where “green-positive” was defined as 1.45 times the local background-subtracted fluorescent signal. After a 2-3-hour lag period post-RSL3 treatment, the cellular green fluorescence signal began increasing. The rate of change in total green-positive area per well from 3–9 hours post-compound treatment was calculated, then expressed relative to that observed in the RSL3-only control treatment group, which was defined as 100%.Q7 cells were cultured in standard culture medium for 18 hours prior to compound addition. After the specified treatment time, the culture medium was removed and adherent cells were washed with PBS. The wash was removed and adherent cells were harvested by adding 1 mL of 100 mg/mL succinic anhydride in 95:5 (acetonitrile:triethylamine) followed by immediate cell scraping. After cell scraping, samples were transferred to a 1.5 mL Eppendorf tube, vortexed for 15 seconds, and kept on wet ice until all samples were harvested. Samples were vortexed again for 15 seconds and centrifuged in a Thermo Sorvall Legend XFR centrifuge at 12,000 rpm, 4°C, for 3 minutes.Following centrifugation, samples were protein-precipitated by addition of 3 volumes of acetonitrile. Following precipitation, samples were vortexed for 30 seconds and centrifuged in a Thermo Sorvall Legend XFR centrifuge at 4000 rpm, 4°C, for 10 minutes. Subsequently, 1 volume of supernatant was transferred to a 384-well injection plate and diluted with one volume of mobile phase A and pipette mixed. Internal standard was added to the 384-well plate and pipette mixed. The injection plate was vortexed and centrifuged for a final time at 4000 rpm, 4°C, for 10 minutes and subsequently placed in a chilled Shimadzu SIL-30AC autosampler at 4°C for LC-MS/MS analysis.αT, αTQ and αTHQ. αT: 430.257→165.1; αT-succinate: 531.368→265.3; αTQ: 429.253→165.1; αTHQ: 431.253→165.1; αTHQ-mono succinate: 572.208→554.1; αTHQ-bis succinate: 666.458→531.4. For concentrations reported: αT = [αT] + [αT-succinate]; αTQ = αTQ; αTHQ = [αTHQ] + [αTHQ-mono succinate] + [αTHQ-bis succinate]. Note: αTHQ and αTHQ-mono succinate were not observed in this experiment.Samples were analyzed using a developed method on a Shimadzu 30AD using micro-flow with a Shimadzu SIL-30AC autosampler, and coupled to an AB Sciex 6500+ QTRAP mass spectrometer. The MS/MS instrument was operated in positive ESI mode. Electrospray conditions for the μLC-ESI-MS/MS method were optimized and were as follows: Ion spray voltage was set to 5500V, temperature of 250°C, curtain gas of 10, CAD gas of 10, and ion source gas 1 and 2 of 15 and 60 psi respectively. Analyzer parameters were optimized for each compound using a combination of manual tuning and compound optimization. The following MRM transitions were used to quantify concentrations of 18  column using a reverse phase gradient mobile phase method was used for chromatographic separation. The composition of mobile phase A was 0.1% formic acid in water. The composition of mobile phase B was 3:1 ). Mobile Phase B was ramped from 30% to 98% over 7 minutes. The total run time was 8 minutes. Data were analyzed using Sciex Analyst Chromatography software, version 1.6.2 and Sciex MultiQuant software. The standard curve equation (y = mx +b) is generated from the calibration standards with weighted regression. Our recent PCT Publication No. WO 2018/081644 also describes the hydroquinone succinate capping methodology in more detail. Intracellular concentrations were estimated assuming spherical cells with an average diameter of 18 μm (estimated using Coulter impedance measurement), with input of 800,000 cells per 1 mL extraction volume.A Phenomenex Kinetex CThis study was approved by the MPI Research IACUC Committee. Animal treatment was in compliance with the U.S. Department of Agriculture’s (USDA) Animal Welfare Act  and performed in an AAALAC accredited facility (File #000243) following The Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy Press, Washington, D.C. The facility maintains an Animal Welfare Assurance statement with the National Institutes of Health Office of Laboratory Animal Welfare.® Sprague-Dawley rats  were dosed once daily for four days by oral gavage to achieve 100 mg/kg dose of d4-α-tocopherol formulated in sesame oil. Four hours after final dose, 0.5 mL of whole blood was collected into Lithium Heparin tubes via the sublingual vein. Blood was centrifuged within 15 minutes of collection and clear supernatant was transferred to 0.2 mL cryotubes. Plasma samples were rapidly frozen and then stored at -80°C until analysis. Euthanasia was performed by carbon dioxide inhalation, after which the tissues were collected and weighed, then chilled PBS was added at a ratio of 3 mL per gram of tissue for homogenization. The homogenate was frozen on dry ice and stored at -80°C until further analysis.Fed male CD4-α-tocopherol and d4-alpha-tocopheryl quinone in plasma and tissues was performed using an Eksigent microLC 200 coupled to a Sciex 6500 Qtrap. Briefly, samples were thawed at 4°C then vortexed. Both plasma and tissue homogenates were protein-precipitated by the addition of ice cold acetonitrile in a 3:1 ratio (ACN:sample). Following precipitation, samples were vortexed for 30 seconds and centrifuged at 4000 rpm for 10 minutes at 4°C. Fifty microliters of supernatant was added to a PCR injection plate containing 50 μL of a 0.1% Formic Acid solution then sealed for mass spectrometry analysis.Quantitation of d4-α-Tocopherol and d4-α-tocopheryl quinone were baseline resolved on a Waters BEH-C18 column . The composition of mobile phase A was 0.1% formic acid in water. The composition of mobile phase B was 0.1% formic acid in 3:1 acetonitrile: isopropyl alcohol. Gradient elution was employed, with mobile phase B being ramped from 20% at 0 to minutes, to 98% at 2 minutes and held for 2.5 minutes. The total run time was 5 minutes. Electrospray conditions for the μLC-ESI-MS/MS method were optimized for a flow rate of 65 μL/min and were as follows: Ion spray voltage was set to 5500 V, temperature of 150°C, curtain gas of 20, CAD gas of 10, and ion source gas 1 and 2 of 20 and 60 psi, respectively. Q1 and Q3 transitions for each analyte and voltage settings  were optimized for each compound using a combination of manual tuning and compound optimization.d4-α-tocopherol and d4-α-tocopheryl quinone in tissue homogenates were determined with μg analyte / g input tissue units, then converted to μM units using the appropriate analyte molecular weights and assuming the following tissue densities . Quinone conversion percentages were calculated using the formula Q / (Q + C) *100 where Q = quinone concentration (in μM) and C = parent chroman concentration (in μM).Concentrations of d® between 0–100 μM in triplicate per concentration was also generated on each assay plate. Diluted compounds and standards were removed from the glovebox and reactions initiated by addition of aerobic fluorescein reagent and radical initiator. Fluorescein fluorescence was monitored at excitation wavelength of 480 nm and emission wavelength of 520 nm every minute for one hour. AUC analysis was performed as described in the kit protocol. The “Trolox equivalents” at each concentration of test compound were calculated and the average Trolox equivalents at the two compound concentrations are presented. The errors were determined by propagating errors from the SEMs of the triplicate determinations to the averages across the two triplicate determinations.ORAC assay kits were purchased from Cell Biolabs. Assays were performed per the manufacturer’s instructions for lipophilic samples with minor modifications. DMSO stock solutions of compounds were diluted in an anaerobic glovebox to final concentrations of 25 and 12.5 μM in triplicate. A standard curve of TroloxαT, αTCC, αTQ or αTHQ in DOPC  were prepared by thin-film hydration followed by extrusion to obtain large unilamellar vesicles at 120 nm in diameter with a polydispersity index (PDI) < 0.1 in DPBS buffer (pH 7.5). PDI measurements were determined using a Malvern Zetasizer Nano-ZSP.Liposomes containing 2 mol% of RTA assays were prepared by diluting liposomes (to 2.5 mM [lipid]) in DPBS buffer and incubating at room temperature with 500 nM BODIPY™ 581/591 C11 dye for 15 minutes. A fresh stock of 20 mM AMVN , Wako Chemicals USA, Inc.) was prepared in MeOH and added to the liposome mix to a final concentration of 2 mM immediately before data collection. The decrease in the reduced dye’s fluorescence emission was measured every 30 seconds at 37°C, using excitation/emission of 575/600 nm with a 590nm cutoff filter, on a Molecular Devices Spectramax M2. The data plots were normalized to 100% fluorescence using the initial time points. The AUC analysis was performed using GraphPad Prism 7.Rabbit 15-lipoxygenase  was obtained from Enzo Life Sciences, Inc. Sodium cholate  was obtained from Sigma-Aldrich. Dihydrorhodamine  was obtained from Biotium. Arachidonic acid  and soybean 15-lipoxygenase  were obtained from Cayman Chemical. The 96 half-well black, flat bottom assay plates  were obtained from Corning, Inc.Enzyme assays  were conducted in Corning 96 half-well black, flat bottom assay plates and contained final concentrations of 50 μM arachidonic acid as a substrate, 1:10 (v:v) cholate mix (2% (w:v) sodium cholate and 2% (v:v) DMSO), 40 μM dihydrorhodamine 123 and 3 U/mL rLOX15 in buffer A . Stock solutions of test compounds were prepared in DMSO and then diluted to final assay concentrations in cholate mix such that final cholate and DMSO concentrations were maintained at 0.2%. Reactions were initiated by addition of 50 μL of enzyme mix (80 μM dihydrorhodamine 123 and 6 U/mL rLOX15 in buffer A) to wells containing 50 μL substrate and cholate mix in Buffer A and linear rates were assessed via fluorescence, using excitation/emission of 500/536 nm, on a Molecular Devices Spectramax M2 every 10 seconds for 5 minutes at room temperature.αTHQ or αT3HQ  from a DMSO stock. All samples contained the same final concentrations of ethanol and DMSO as sample 3. The EPR spectra of buffer alone were also evaluated to allow for baseline subtraction.Lipoxygenase: Soybean 15-lipoxygenase (SLO) was buffer exchanged into 50 mM borate buffer, pH 9.0 containing 10% (v/v) glycerol. The resulting protein solution was concentrated to approximately 100 μM, based upon the supplier’s activity per mL and specific activity per mg. The active-site iron oxidation state was further evaluated by EPR under three treatment conditions: 1) No treatment, 2) oxidation with 3-fold molar excess of lipid hydroperoxide  from an ethanol stock, 3) oxidation with 3-fold molar excess of lipid hydroperoxide followed by 10-fold molar excess of CW-EPR spectra were recorded at 150 K on a Bruker ELEXYS E540 spectrometer fitted with an X-band microwave bridge, a Super High Sensitivity Resonator (SHQ), and a Nitrogen VT apparatus. The spectra were recorded at 9.43 GHz with 1.002 mW microwave power, 10 G modulation amplitude, 100 kHz modulation frequency, 25 scans.CW-EPR spectra were recorded at room temperature in ambient air on a Bruker EMXnano spectrometer fitted with an X-band microwave bridge. All spectra were recorded at 9.61 GHz with 10 mW microwave power and 5 G modulation amplitude, 100 kHz modulation frequency, 25 scans.Ferroptosis was initiated by RSL3  in Q7 cells as described above. The protective activity of co-treated compounds was assessed also as described above. Four to five hours after RSL3 treatment, the conditioned medium was collected into 1.2-mL cluster tubes containing ice-cold acetonitrile at a ratio of 1:2. Tubes were snap frozen on dry ice and stored at -80°C.Prior to analysis, samples were thawed on wet ice and then vortexed briefly. Samples were centrifuged at 4000 rpm for 15min at 4°C. Supernatant (40 μL) was transferred to a 96-well injection plate and 10 μL of both internal standard and HPLC grade water were added. A Shimadzu Nexera MP system coupled to a Sciex 6500 Qtrap and configured for micro-flow was used to analyze all samples. Samples were maintained at 4°C in the Nexera MP autosampler for the duration of the analytical run.8 column  operated at 45°C was used for chromatographic separation. The total run time of the liquid chromatography method was 10 min. Mobile phase A consisted of water with 0.1% formic acid. Mobile phase B was composed of 0.1% formic acid in 3:1 acetonitrile:isopropyl alcohol. Gradient elution was employed, with mobile phase B being ramped from 5% at 0min, to 50% at 2 min, and finally 98% at 7.5 min. Electrospray conditions for the μLC-ESI-MS/MS method were optimized for a flow rate of 125 μL/min and were as follows: Ion spray voltage was set to -4500 V, temperature of 250°C, curtain gas of 20, CAD gas of 10, and ion source gas 1 and 2 of 20 and 40 psi, respectively. Analyzer parameters were optimized for each compound using a combination of manual tuning and compound optimization. The following MRM transitions were used to quantify concentrations of 15-HETE, 12-HETE and Arachidonic Acid (AA). 15-HETE: 319→219; 12-HETE: 319→179; AA: 303→259.A Thermo Gold-Ct-test with Welch’s correction was used. Statistical significance throughout was defined as p<0.05.All statistical analyses were performed using GraphPad Prism v.7.03 for Windows. If all groups showed apparently normal distributions (by Shapiro-Wilk test), then one-way ANOVA tests were performed, with Sidak’s correction for multiple comparisons applied when appropriate. If even one group did not meet normality assumptions, then a non-parametric Kruskal-Wallis test was used, with Dunn’s multiple comparisons test applied when appropriate. When only comparing two groups, then an unpaired αT) are responsible for its previously reported anti-ferroptotic activity S,3R)-RSL3, a well-described irreversible inhibitor of GPX4, to induce ferroptosis in Q7 cells. RSL3 treatment of Q7 cells resulted in dose- and time-dependent loss of cell viability. Typically, at RSL3 concentrations ≥1 μM, 80–95% loss of cell viability was observed 18 h after RSL3 treatment, with onset of cell death observed ~5 h after RSL3 addition. Consistent with published reports, αT prevented RSL3-induced cell death with an EC50 of 5.2 ± 2.8 μM  than αT itself  ,9,24–26,αTQ and αTHQ metabolites compared to the parent αT led us to assess the prevalence of conversion in biological systems. To demonstrate in vivo the oxidative chroman ring opening of αT to αTQ, we synthesized stable-labelled d4-αT and dosed fed male rats once daily for four days by oral gavage at 100 mg/kg  in the small intestine, reaching concentrations of approximately 200 μM, to plasma where the (d4-αT:d4-αTQ) ratio was greater than 100:1. Due to the rapid and spontaneous re-oxidation of the hydroquinone (αTHQ) to the quinone (αTQ) .–36.αTQ aαT and its metabolites, αTQ and αTHQ, in our cellular ferroptosis model system, we established an LC-MS/MS method to quantitate all three analytes simultaneously in cell extracts. Because αTHQ is rapidly oxidized back to αTQ upon exposure to atmospheric oxygen, [bis-succinate ester [αT, αTQ, and αTHQ  in the culture medium containing 10% fetal bovine serum. After αT supplementation  in the Q7 cell medium, the cellular αT, αTQ, and αTHQ concentrations were each elevated H:quinone oxidoreductase 1 (NQO1) [αTQ , the cellular exposure of αT was unaffected  is reduced to the hydroquinone (αTHQ) form readily in cells; indeed, the αTHQ exposures exceed those of αTQ by 3- to 4-fold in all cases tested.To assess directly the cellular exposure of  oxygen,  we quantte ester . We obseelevated , yet mai1 (NQO1) ,39. When 1:22:76 , a markeαT chroman ring to the corresponding αTQ quinone is necessary for anti-ferroptotic activity, we designed and synthesized a novel non-metabolizable analog of αT we refer to as αT carbochroman . We confirmed that, similar to αT, αTCC forms a radical through hydrogen atom donation, and this radical is delocalized over the carbochroman ring system  of αT, αTCC, αTQ, and αTHQ in liposome vesicles as measured by an increased lag period prior to quenching of BODIPY™ 581/591 C11 fluorescence (αT> αTCC>> αTHQ> αTQ (αT and αTCC have similar activity and are between 2.5–4 fold greater than αTQ and αTHQ (vs. closed bars), and argues that radical trapping antioxidant activity alone is not sufficient to explain the increased potency of αTQ and αTHQ , suggesting that free fatty acids might be released during this death process , while αT, αTCC, and αTQ all failed to decrease enzyme activity by more than 20% at 100 μM, the highest concentration tested , and EPR spectroscopy to measure its redox state [3+ state, which was spin quantified and set to 100% Fe3+  indicated that Fe3+ reduction by αTHQ was specific to the enzyme-bound metal.The catalytic activity of 15-Lipoxygenase requires a non-heme iron center to be in the Feox state ,45. Incu00% Fe3+ . Upon adet al., [αT3 was more potent in inhibiting ferroptosis than αT  of alpha-tocotrienol quinone αT3Q, together with a favorable pharmacokinetic and safety profile, position it as an attractive candidate to treat diseases involving oxidative stress, inflammation, and ferroptosis[To determine the key molecular features of vitamin E and its metabolites that afford the ability to inhibit 15-LO and ferroptosis, we prepared a library of each of the naturally occurring vitamers  and their major metabolites . With thet al.,  we obserSLO Figs . The potrroptosis.αT) is responsible for its anti-ferroptotic activity, we used the novel, non-metabolizable αT analog, αTCC, as a chemical biology probe and found that the radical trapping activity of αT is insufficient to explain its anti-ferroptotic cellular activity  in rm αTHQ; ; deactivrm αTHQ;  by αTHQ 2+ state ; (4) the2+ state  and 6A a2+ state .αT) and vitamin E quinone (αTQ) possessed protective activity in fetal resorption studies[αT chroman) was defined as the essential nutritional factor, not the vitamin E quinone. More recently, a vitamin E effect on fetal resorption was demonstrated in mice by action at the 15-LO/GPX4 ferroptotic pathway[In 1939, Evans and Bishop noted that both vitamin E Click here for additional data file.S2 Method(DOCX)Click here for additional data file.S3 Method(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


By using a one-pot microwave-assisted synthesis route in 1.6 M KOH (MnVII:MnII = 0.33), a pure layered δ-MnO2birnessite phase without any hausmannite traces was obtained after only a 14 h reaction time period at 110 °C. Attempts to enhance crystallinity level of as-prepared birnessite through increasing of reaction time up to 96 h in 1.6 M KOH failed and led to decreases in crystallinity and the emergence of an additional hausmannite phase. The influence of MnII:OH− ratio (1:2 to 1:10) on phase crystallinity and hausmannite phase formation for 96 h reaction time was investigated as well. By increasing alkalinity of the reaction mixture up to 2.5 M KOH, a slight increase in crystallinity of birnessite phase was achieved, but hausmannite formation couldn’t be inhibited as hoped. The as-prepared layered δ-MnO2 powder material was spray-coated on a carbon paper and tested in laboratory cells with Al or Zn as active materials. The Al-ion tests were carried out in EMIMCl/AlCl3 while the Zn-Ion experiments were performed in water containing choline acetate (ChAcO) or a ZnSO4 solution. Best performance in terms of capacity was yielded in the Zn-ion cell (200 mWh g−1 for 20 cycles) compared to about 3 mAh g−1 for the Al-ion cell. The poor activity of the latter system was attributed to low dissociation rate of tetrachloroaluminate ions (AlCl4−) in the EMIMCl/AlCl3 mixture into positive Al complexes which are needed for charge compensation of the oxide-based cathode during the discharge step.This work reports on the synthesis of layered manganese oxides (δ-MnO Therefore alternative electrochemically-active and abundant materials such as Na, Mg, Mn, Al, and Zn are appealing candidates. However, intercalation of multivalent ions into common host matrixes such as manganese oxide spinel is strongly affected by higher diffusion barriers than monovalent ones . Nonethe−1 vs. 2 Ah L−1 for LIB), its well established manufacturing and recycling processes, as well as its global availability and abundance. Thus, the AIB covers all prerequisites to develop a sustainable and ecologically-benign battery. Historically, this concept was limited due to the lack of electrolytes for reversible aluminum deposition and dissolution − complex with a stability constant value of 3.98 × 1016. Hence, once the solution reaches the OH− saturation level, the complex is in equilibrium with its precipitate Mn(OH)2.Reactions (6) and (7) are favored due to negative free enthalpies, and therefore, an essential indicator for assumed first reaction steps during MnO2 can further react with MnO4− under formation of MnO42− as described before in Reaction (7). Manganate(VI) MnO42− subsequently reacts with Mn(OH)2 under the formation of β-MnOOH and blue-colored hypomangante(V) MnO43− as followsReformed Mn(OH)0 = 0.96 V (vs. NHE) and reacts with β-MnOOH (E0 = 0.95 V) to birnessite type δ-MnO2 according to Equation (10):Hypomanganate has a high oxidation potential of E4− (E0 = 0.558 V) and MnO42− (E0 = 0.60 V) in alkaline media are not high enough for MnOOH oxidation, the limiting reaction step is the formation of hypomanganate(V), despite relative high exothermic reaction enthalpy value (−234 kJ mol−1). In our experiments at OH− = 0.6 and 0.8 mol L−1, other manganese oxides like hausmannite (Mn3O4) were yielded (4 of Equation (9) increases with higher [OH−] concentration. This assumption is supported by the increase in crystallinity of formed birnessite at 1.6, and especially, 2.5 mol L−1  of MnO2 formation explain the extremely long reaction time of several days at room temperature reported by Luo et al., and at least 14 h at 110 °C in microwave synthesis in this work.The law of mass action for Equations (9) and (10) and the calculated endothermic reaction enthalpy (+106 kJ mol−] and [MnO42−] (Equation (11)). Due to low hypomanganate, and therefore, low MnO2 concentration at 0.6 & 0.8 mol L−1 hydroxide, the equilibrium is massively shifted to the educt site (Equation (12)). This might explain the formation of crystalline birnessite phase MnO2 at [OH−] concentrations ≥ 1.6 mol L−1. The formation of hausmannite at [OH−] concentrations ≤ 1.6 mol L−1 can be explained by the thermodynamically-favored reaction of residual Mn(OH)2 with MnOOH (−46 kJ mol−1) according to Reaction (13). The presence of residual Mn(OH)2 can be explained by significantly-decreased hypomanganate formation, according to Equation (11). After 96 h of heating, the thermodynamically more stable hausmannite structure is formed due to the reduction and collapse of δ-MnO2.As assumed, the formation of hypomanganate is directly correlated to the concentration of [OHhypomanganate MnO43− formation that is directly dependent on the MnO42− and [OH−] concentration. Our experiments confirm that strongly-alkaline environments between 2.5 and 0.8 with an optimum at 1.6 mol L−1 and elevated temperatures are required for the fast formation of layered δ-MnO2. However, higher temperatures than 110 °C lead to the formation of hausmannite due the collapse of the birnessite crystal structure.The limiting reaction step is the 2 and hausmannite , combined with 2 h equilibration time at respective temperatures of up to 600 °C.In-situ XRD measurements were carried out to evaluate thermal material stability, and to determine optimal drying parameters for removing all interlayer water, before it can be assembled to an aluminum-ion battery together with the highly water-sensitive electrolyte EMIMCl/AlClbirnessite polymorph KxMnO2 with x ≤ 0.5. Thus, the as synthesized δ-MnO2 does not fit perfectly with the reference spectra of hydrated or dried single phase monoclinic δ-MnO2  profiles of synthesized δ-MnO−1 which corresponds to ~0.8% of theoretically value (524 mWh g−1) based on Mn+3/+4O2 redox pair  has to dissociate into Al3+ (ø = 1.08 Å) to enter the gallery. We conclude that the central Al3+ ion is too strongly shielded by four Cl− anions in the tetrahedral configuration. Consequently, the affinity of AlCl4− anions for dissociation into a positive charge species, which is needed for charge compensation of the cathode material during reduction step, is very poor.As determined in 3+ ion intercalation into a vanadium oxide (VO2) tunnel structure that showed significantly higher capacities in EMIMCl/AlCl3 (116 mAh g−1 @ 0.9–0.1 V vs. Al/Al3+ for 100 cycles) [−1 with a V2O5 cathode can be attributed to the presence of fully dissociated Al3+ ions in the electrolyte [3+ ions in the electrolyte and/or oxide matrix was provided by e.g., Raman, NMR or XPS analysis, respectively.In contrast to our oxide cathode, W. Wang et al. reported successful reversible Al cycles) . Howeverctrolyte . In both4− ions in the AIB electrolyte, fully dissociated Zn2+ ions (ø = 1.48 Å) are present in aqueous electrolytes. Additionally, as shown in 2 has an increased interlayer distance of 7.1 Å, resulting in an accessible gallery height of ~3.8 Å. Thus, the aqueous ZIB appeared to be a priori a more suitable system to demonstrate the MnO2 ability for multivalent ion intercalation.In contrast to the AlCl2 in choline/zinc acetate and zinc sulfate electrolytes is shown in 2+/Mn3+. Above 1.6 V, the current starts to increase rapidly, indicating the deintercalation of H+ and Zn2+, as well as possible water splitting. The corresponding cathodic peaks shifted between 1.37–1.5 V. An unidentified small reduction peak was observed at 1.6–1.7 V. The overall specific current is significantly higher compared to that of the AIB (0.8–1.8 A g−1 vs. 50–110 mA g−1). Interestingly, in contrast to AIB-CVs in 2 matrix.Influence of a potential window on the electrochemical behavior of δ-MnO3+/2+ visible in choline acetate electrolyte  values of Zn2+ from MnO2 matrix [−1 at 1.6 V, which may be related to a higher zinc concentration in sulfate solution. The blurred potential above 1.68 V might be explained by oxygen evolution due to water decomposition. The change in CV shape between the 1st and 4th curves indicates a structural change in MnO2 matrix . Because of electrolyte decomposition and poor dissociation of AlCl4− into positively-charged species, EMIMCl/AlCl3 appeared to be an inappropriate electrolyte for high-energy AIB batteries with metal oxide cathodes.In order to allow easier comparison between AIB and ZIB cyclic voltammograms, redox peak potential regions were normalized to the standard hydrogen reference electrode (SHE), and listed in .7 V see , whereas2 as the cathode material are shown in −1 with an energy efficiency ≥ 83%. This specific energy corresponds to about 38% of the theoretical value of pure MnO2 equal to ~527 mWh g−1 by assuming a one-electron transfer step based on the Mn4+/3+ redox reaction. The mean discharge voltage was around 1.48 V. The discharge curve shows a plateau region till 1.38 V. As published by Sun et al. [+ insertion (120–140 mWh g−1) from the slightly acidic electrolyte (pH~4.7). Afterwards, the discharge curve became significantly steeper due to insertion of bigger Zn2+ ions into MnO2 matrix (70–80 mWh g−1) at ≤1.35 V. This voltage value is in very good accordance with measured ≈1.3 V intercalation potential as shown in 2 matrix compared to that of protons. Since the ionic radii of K+ (138 pm) and H2O (2.78 Å) defines the interlayer distance in δ-MnO2 matrix, hydrogen (53 pm) and zinc (74 pm) can be a-priori intercalated. The gravimetric energy and power density was determined to 210 Wh kg−1 (748 Wh L−1) and 44.4 W kg−1. After the 20th cycle, the capacity started to decrease continuously down to 72 mWh g−1 after the 80th cycle (−1 (392 Wh L−1) which is ~48% less than that yielded during the first cycle.Results from galvanostatic charge & discharge experiments of zinc-ion cell with δ-MnOn et al. , this flth cycle b. After 2birnessite phase with hausmannite Mn3O4 as side phase. The optimum KOH concentration in terms of hausmannite impurities was found to be 1.6 mol L−1. Further efforts to reduce side-phase were very successful by only reducing reaction time down to 14 h. Experiments with shorter reaction time failed. These should be eventually performed at slightly higher temperature than 110 °C.A modified microwave-assisted hydrothermal route based on comproportionation of potassium permanganate and manganese nitrate at 110 °C within 96 h led to layered δ-MnO4− in EMIMCl/AlCl3 electrolytes which does not fit into the gallery of dried MnO2 (d = 3 Å). It must be concluded that dissociation of the AlCl4− into the much smaller Al3+ ions (d = 1.08 Å) didn’t take place due to the strong shielding by chloride ions. In ZIB, the fully-dissociated Zn2+ ions (d = 1.48 Å) are readily able to intercalate into the hydrated interlayer space (d = 3.8 Å). A specific discharge energy of about 200 mWh g−1MnO2 at 1.48 V mean voltage and 85% energy efficiency was obtained within first 20 cycles in 1 M ZnSO4. At C5 discharge current, capacity is induced by first proton intercalation between 1.65 and 1.3 V, followed by Zn intercalation at lower cell polarization values. Thus, the as-synthesized δ-MnO2 is active for divalent-ion intercalation, and is a very promising candidate as cathode material in Zn-ion batteries. However, cyclability and cathode material conductivity and stability have to be significantly improved.The as-synthesized manganese oxide powders have been tested as cathode material for both Al-ion and Zn-ion battery. In AIB, a very poor capacity was yielded due to large size (ø = 5.5 Å) and negative charge of AlCl


Rotaviruses (RVs) are a major cause of acute children gastroenteritis. The rotavirus P [10] belongs to P[I] genogroup of group A rotaviruses that mainly infect animals, while the rotavirus P [10] was mainly identified from human infection. The rotavirus P [10] is an unusual genotype and the recognition pattern of cellular receptors remains unclear.We expressed and purified the RV P [10] VP8* protein and investigated the saliva and oligosaccharide binding profiles of the protein. A homology model of the P [10] VP8* core protein was built and the superimposition structural analysis of P [10] VP8* protein on P [19] VP8* in complex with mucin core 2 was performed to explore the possible docking structural basis of P [10] VP8* and mucin cores.Our data showed that rotavirus P [10] VP8* protein bound to all ABO secretor and non-secretor saliva. The rotavirus P [10] could bind strongly to mucin core 2 and weakly to mucin core 4. The homology modeling indicated that RV P [10] VP8* binds to mucin core 2 using a potential glycan binding site that is the same to P [19] VP8* belonging to P[II] genogroup.Our results suggested an interaction of rotavirus P [10] VP8* protein with mucin core 2 and mucin core 4. These findings offer potential for elucidating the mechanism of RV A host specificity, evolution and epidemiology. Reoviridae family, possesses 11 segments of double-stranded RNA encoding 12 proteins, including six structural proteins  and six nonstructural proteins (NSP1 to NSP6). Based on the sequence variability of the outer capsid glycoprotein VP7 and protease-sensitive spike protein VP4, group A rotavirus, the most common species, is classified into various G and P types respectively. The current evidence indicates that there exists at least 27 G and 37 P genotypes  is an unusual genotype, which was first identified in a human G8 RVA strain , 7. Sinc is an unThe first step of rotavirus infection involves the recognition of specific cell surface glycans in the initial cell attachment step. Recent studies indicate that RVs recognize histo-blood group antigens (HBGAs) as potential receptors –15. The Esherichia coli strain BL21 (DE3) and then the expression of protein was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.4 mM at 22 °C for 16 h. The recombinant protein was purified using Glutathione Sepharose 4 Fast Flow  as reported previously [g. The VP8* protein (amino acids 1 to 230) of human P [19] Mc345 (GenBank: D38054) was conserved in our lab [The VP8* gene segment from a human P [10] strain, RVA/Human-wt/IND/mcs60/2011/G3P [10] (GenBank accession number JQ358765.1), was synthesized by Genewiz Company . The full VP8* gene (encoding amino acids 1 to 232) was cloned with an N-terminal glutathione S-transferase (GST) tag into a pGEX4T-1 expression vector. The vector was transformed into A panel of saliva samples with typed A, B, and O and secretor status was kept in our laboratory. HBGA phenotypes of these saliva samples have been determined by enzyme immunoassays as previously described . The sala], Leb, Lec, Lex, Ley, A, B, H1, H2, H3, αaβ1,4, mucin core 2, mucin core 4, mucin core 6, Neu5Ac, Neu5Gc) (GlycoTech) were added at 0.2 μg per well at 4 °C overnight. Then, HRP-conjugated streptavidin (Abcam) was added at 0.1 μg per well at 37 °C for 1 h. At each step, the plates were washed five times with 0.5% PBS-Tween 20 buffer. The plate was added with 100 μl TMB Substrate Solution and incubation in the dark for 15 min. Finally, 100 μl Stop Solution were added to each well, and the absorbance at 450 nm was determined within 5 min.The purified GST-VP8* fusion proteins of human P [10], P [19], P [14] and GST protein were diluted with PBS at 200 μg/ml separately. The 96-well microtiter plate was coated with the diluted GST-VP8* at a volume of 100 μl per well at 4 °C overnight and then blocked with 5% nonfat milk, as reported previously , 20. Synhttp://swissmodel.expasy.org/). Superimposition structural analysis of P [10] VP8* protein on P [19] VP8* in complex with core 2 (PDB identifier [ID] 5VKI) was performed using the PyMOL software package (https://pymol.org/2/).A homology model of the P [10] VP8* core protein was built based on the X-raycrystal structure of the rhesus rotavirus VP4 sialic acid binding domain in complex with 2-O-methyl-alpha-D-N-acetyl neuraminic acid (SMT ID: 1kqr.1) as the template by SWISS-MODEL automated protein structure homology modeling server . Informed consent was obtained from the members who provided samples. All experiments were performed in accordance with relevant guidelines and regulations of China.E.coli, and the molecular weight was ~ 46 kDa . The GST-VP8* fusion protein was expressed in soluble form in 0 was expWe first measured the binding patterns of P [10] VP8* to A-, B-, and O-type saliva. The purified GST-VP8* protein was tested in a saliva binding assay using saliva samples with known A, B, O secretors and non-secretors types. The saliva samples from 214 individuals were collected in Guangdong province including 53 individuals with A-type, 36 individuals with B-type, 9 individuals with AB-type, 106 individuals with O secretors type, and 10 individuals with non-secretors type. Human P [10] VP8* protein showed binding to saliva of all A, B, AB, O secretors and non-secretors types and GST protein did not reveal binding activity as negative control  as the template and the sequence identity is 74.21% Fig. . RecentlGroup A rotaviruses are a major cause of acute childhood diarrhea. The human P [10] rotavirus strains were in association with strains of various genotypes. So far, few isolates of rotavirus P [10] have been reported in the literature. The molecular evolution of rotavirus P [10] remains unclear. The characterization of recognition patterns of P [10] with cellular glycans is required to elucidate the origin of RV P [10] and understanding on the epidemiology of the virus. In this study, We performed different assays to explore the receptor binding specificity of P [10] RV VP8*, including the oligosaccharide binding assay, saliva-based binding assay, and structural modeling.Significant advancements have been made in elucidating RV and host interactions. Recent reports discovered that RVs recognize HBGAs as attachment factors or receptors. HBGAs are carbohydrates that distribute abundantly on mucosal epithelia of the body, and as soluble oligosaccharides in most body fluids except cerebrospinal fluid. HBGAs are polymorphic with different ABO, Lewis and secretor versus non-secretor types. RV-HBGA interactions correlate with the host susceptibility of RV infection and illness. RVs P [8] and P [4] preferably infect individuals who were secretors/partial secretors . DistincMucins are the main structural components of mucus. Mucin core 2 and mucin core 4 are commonly found in intestinal mucins. In humans, gastric and duodenal mucins generally contain the core 1  and the core 2  GalNAcα1-Ser/Thr) structures, and the core 3  and core 4  GalNAcαSer/Thr) structures are predominant in the colon . Mucin gThe binding characterization of RV and cellular receptors plays a role in the cross-species transmission of RVs. The P 10] genotype belongs to the P[I] genogroup of group A rotaviruses. Most of RV genotypes in genogroup P[I] mainly infect animals, while the rotavirus P [10] strains were mainly identified from human infection –10, 29.  genotypeHuman rotavirus P [10] VP8* protein bound to all ABO secretor and non-secretor saliva and could bind strongly to mucin core 2 and weakly to mucin core 4. P [10] VP8* protein binds to mucin core 2 using same potential glycan binding site with that of P [19] VP8* protein.


The dataset is made publicly available and can be useful for extending the structural and bioactive research and critical analyses of lignin and LCC.The data presented in this article are related to the research article entitled “Structure-antioxidant activity relationship of active oxygen catalytic lignin and lignin-carbohydrate complex” . It supplements the article with thermostability of milled wood lignin (MWL) and alkali-oxygen lignin (AOL), main substructures of lignin in rice straw, main products and yield of nitrobenzene oxidation of lignin-carbohydrate complexes (LCCs), Fourier transform infrared spectroscopy of LCCs, radical (ABTS·) scavenging ability of lignins and signal assignment of lignins and LCCs in nuclear magnetic resonance spectra ( After grinding and tabletting, the FTIR spectra was recorded with the scan resolution of 4 cm2.21H and 13C NMR. 20 mg of acetylated lignins was dissolved in 0.5 mL DMSO-d6 for 1H NMR detection. For the quantitative 13C NMR experiment, acetylated lignin (150 mg) was dissolved in DMSO-d6 (0.5 mL). Chromium (III) acetylacetonate  was added to provide complete relaxation of all nuclei. The mixture was then transferred to a Shigemi microtube and characterized at 25 °C. The acquisition parameters were: 90° pulse width, a relaxation delay of 1.7 s, and an acquisition time of 1.2 s. A total of 20,000 scans were collected.MWL and AOL were acetylated according to the method reported by Lu and Ralph d6. The number of collected complex points was 2048 for the 1H-dimension with a recycle delay of 1.5 s. The number of transients was 64, and 256 time increments were recorded in the 13C-dimension. The 1JCH used was 145 Hz. Processing used typical matched Gaussian apodization in the 1H-dimension and squared cosine-bell apodization in the 13C-dimension. Prior to Fourier transformation, the data matrices were zero-filled to 1024 points in the 13C-dimension.For 2D HSQC NMR test of LCCs, the LCC samples (50 mg) were dissolved in 0.5 mL of DMSO-2.3Nitrobenzene oxidation was applied to the LCCs according to the procedure reported by Chen 2.4−5 mol/L. ABTS· was generated by reacting 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (7 mM) with 2.45 mM potassium persulfate (K2S2O8) in ultrapure water and then letting the solution stand for 15 h in the dark at room temperature. The radical solution was adjusted to obtain an UV absorbance of 0.70 ± 0.02 at 517 nm and 734 nm for DPPH· and ABTS·, respectively. The concentration of lignin and LCCs in tested sample is 0.03 mg/mL. The absorbance of tested sample was measured using a microplate spectrophotometer . The radical scavenging ability was calculated using the following formula:i is the absorbance of the tested sample; Aj is the absorbance of the blank sample via anhydrous ethanol replacing DPPH· or ultrapure water replacing ABTS· solution; A0 is the absorbance of the blank sample via anhydrous ethanol or ultrapure water replacing lignin solution.The DPPH· and ABTS· radical scavenging assay of lignins and LCCs was performed using a spectrophotometric method. Samples were dissolved in 90% 1,4-dioxane/water (v/v). The DPPH· was dissolved in anhydrous ethanol with the concentration of 6 × 10


Panax ginseng, ginsenoside Rg1 has certain anti-aging effects, improving the activity of learning and memory. Studies have showed that ginsenoside Rg1 improves the memory impairment associated with Alzheimer’s disease (AD). In this study, the effects of ginsenoside Rg1 were investigated through the activity of toll-like receptor (TLR) 3, TLR4 and their signaling transduction pathways in amyloid β peptide 25–35 (Aβ25–35) induced AD cell model. Thus we investigated several critical components of the TLR pathway. The neuroglial cell line NG108-15 was stimulated with or without Aβ25–35, while different concentrations of ginsenoside Rg1 were administered. After 24 h, tumor necrosis factor-α (TNF-α), interferon-β (IFN-β) in cell supernatant and inducible nitric oxide synthase (iNOS) in cell lysate supernatant were measured with enzyme-linked immunosorbent assays (ELISAs). The mRNA and protein expression of TLR3, TLR4, nuclear factor kappa B (NF-κB) and tumor necrosis factor receptor-associated factor-6 (TRAF-6) were detected by real-time PCR and western blot methods, respectively. The experimental results showed that Aβ25–35 could markedly raise the level of TNF-α, IFN-β and iNOS, and increase the expressions of mRNA and TLR3, TLR4, NF-κB and TRAF-6 protein in the NG108-15 cells. At the same time, the ginsenoside Rg1 significantly reduced the expressions of proteins and mRNA of TLR3, TLR4, NF-κB and TRAF-6, and down-regulated the levels of TNF-α, IFN-β of cell supernatant and iNOS of cell lysate supernatant in a concentration-dependent manner. In conclusion, ginsenoside Rg1 has good activity for suppressing the signaling transduction pathway of TLR3 and TLR4, and decreasing the inflammation factors induced by Aβ25–35 in NG108-15 cells, and this may be the mechanism of ginsenoside Rg1 action in AD treatment, but more studies are needed to identify its specificity.As one of the most important components of  As is ketc. . In addietc. . Neuroinetc. . Activatetc. . There i [et al.  found thToll-like receptors (TLRs), are essential signaling components of the mammalian host defense system. At least 12 TLRs were found in rats and 10 TLRs in human beings . In AD pPanax ginseng is a well-known Chinese herb used for treating neurodegenerative diseases such as AD [Panax ginseng, can fight against aging, promoting the ability of learning and memory in the normal brain [Chinese herbs from medicinal plants show certain effects for treating AD . Panax gch as AD . Ginsenoal brain . In addial brain ,19. Howe25–35 stimulation . Ginsenoside Rg1 treatment (2 µg/mL) did not affect the levels of TNF-α, IFN-β and iNOS (p> 0.05). However, 4 to 32 µg/mL of Rg1 treatment significantly decreased the production of TNF-α, IFN-β and iNOS in a concentration-dependent manner, although ginsenoside Rg1 at 4 µg/mL did not affect IFN-β level . Treatment with ginsenoside Rg1, markedly reduced the TLR3, TLR4, NF-κB and TRAF-6 mRNA expressions induced by Aβ in a concentration-dependent manner (vs. Aβ stimulated group). Ginsenoside Rg1 (4 µg/mL) did not affect TLR3 expression (p > 0.05), but significantly decreased the expression of TLR4, NF-κB and TRAF-6  of Aβ-stimulated cells. Ginsenoside Rg1 at 8, 16 and 32 µg/mL concentrations inhibited the mRNA up-regulated mRNA expression of TLR3, TLR4, NF-κB and TRAF-6    of Aβ-st25–35 stimulation induced significant elevations of protein expressions of TLR3, TLR4, NF-κB and TRAF-6 . Ginsenoside Rg1 treatment significantly decreased the up-regulated protein expressions of TLR3 and TLR4 which were induced by Aβ in a concentration-dependent manner, although 2 µg/mL ginsenoside Rg1 treatment did not affect the protein levels of TLR3 and TLR4 , which plays a crucial role in initiating immunity against invading microbial pathogens. Each TLR recognizes distinct pathogen-associated molecular patterns (PAMPs) that include lipids, nucleic acids lipoproteins, and proteins. TLRs expressed in a variety of mammalian immune cell types such as NK cells, monocytes, macrophages, dendritic cells, mast cells, neutrophils and endothelial cells ,27. TLRsAll TLRs are located in the cell membrane and have a common expression in the cytosolic domain. Binding specific TLR ligands leads to the recruitment and activation of adapter molecules, such as myeloid differentiation factor 88 (MyD88) which is activated by all TLRs except TLR3 . MyD88-dWe found that normal NG108-15 cells slightly expressed TLR3 and TLR4 mRNA and protein. However, the mRNA and protein levels of TLR3 and TLR4 were significantly elevated when Aβ was added to the cells. Similar changes were also observed in the expressions of NF-κB and TRAF-6, the results of which indicated that TLR3 and TLR4 signaling transduction pathways participated in the inflammation reaction induced by Aβ. The up-regulated expressions of TLR3, TLR4, NF-κB and TRAF-6 were significantly attenuated when the cells were treated with Rg1, indicating that ginsenoside Rg1 might affect TLR3, TLR4 and their signaling transduction pathways.25–35 up-regulated expressions of TLR3, TLR4, NF-κB and TRAF-6, and elevated the levels of TNF-α, IFN-β and iNOS in NG108-15 cells. This study suggests that the TLR3 and TLR4 transduction pathways participate in the inflammation process in the AD cell model. Ginsenoside Rg1 inhibited the up-regulated expressions of TLR3, TLR4, NF-κB and TRAF-6 both at mRNA and protein levels, and decreased the amount of TNF-α, IFN-β and iNOS. The therapeutical effect of ginsenoside Rg1 on AD might be related to TLR3, TLR4 and their signaling transduction pathways.In conclusion, the present study demonstrated that AβIn this paper, we emphasized the influence of Aβ on inflammatory factors, TLR3/4 and their signaling transduction pathways. In our preliminary tests, we investigated the influence of Aβ on cell survival rate, but didn’t examine the influence on cell viability. Whether ginsenoside Rg1 influences the viability of cells and other TLRs signaling pathways or not remains unclear and needs further investigation.Ginsenoside Rg1 was purchased from the National Institute for the Control of Pharmaceutical and Biological Products , DMEM media and fetal bovine serum (FBS) were purchased from Gibco . Primers of TLR3, TLR4, NF-κB, TRAF-6 and β-actin were synthesized by Invitrigen Life Technology Co. Ltd. , and M-MLV Reverse Transcriptase was obtained from Promega  Biotechnology Co. Ltd. , and primary and second antibodies of TLR3, TLR4, NF-κB, TRAF-6, β-actin were produced by Santa Cruz . TNF-α, INF-β and iNOS ELISA kits were purchased from R&D Co., Ltd. . All other chemicals were obtained from Sigma-Aldrich Chemical Co. , unless otherwise stated.3 (2 g/L). Cells were maintained in a humidified 37 °C incubator with a 5% CO2 and 95% air and used for assays during the exponential growth phase.Neuroglial cell line NG108-15 obtained from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences  was cultured in DMEM media supplemented with 10% (v/v) heat inactivated fetal calf serum, penicillin (100 U/L), streptomycin (100 mg/L) and NaHCO25–35  at 2 × 105 cells/mL, and ginsenoside Rg1 was added to the cells at final concentrations of 2, 4, 8, 16 and 32 µg/mL, respectively. In the control group, cells were treated with vehicle alone. After incubation for 24 h, the cell supernatant was collected, and levels of TNF-α, IFN-β were measured with ELISA kits. Then the cells were lysed and centrifuged, and the supernatant of cell lysate was obtained and iNOS was measured with the ELISA method. In another experiment, the cells were harvested, and the mRNA and protein levels of TLR3 and TLR4 were analyzed by real-time PCR and western blot, respectively. The concentration of Aβ25–35 in 5 µmol/L has been reported to reduce cell survival and increase acetylcholinesterase (AchE) activity in NG108-15 cells [Cells were stimulated with or without Aβ15 cells .TNF-α, IFN-β and iNOS Assay After 24 h incubation, TNF-α and IFN-β in the medium and iNOS in cells were measured by ELISA with an R&D System ELISA kit as described previously . In brieThe total RNA was extracted from NG108-15 cells using the TRIzol method as described previously . Briefly−ddCt) as described by Schmittgen et al. [All primers were synthesized by Invitrogen . Real-time PCR assay was performed on a ABI 7300 Real-time PCR meter . All reactions were performed in a total of 20 µL including 1× IQ SYBR Green Supermix from Takara , 250 nM of each primer and 2 µL cDNA as template. PCR conditions were as follows: 95 °C for 30 s to activate the iTaq polymerase, 45 cycles of 5 s at 95 °C and 31 s at 60 °C. Melt curve was performed for all samples in which only sharp melting points were observed, indicating a specific signal and no primer dimers or mispriming. Relative contents of the TLR3, TLR4, NF-κB and TRAF-6 genes were calculated using the ddCt method  to cells monolayers. Insoluble cell fractions were then pelleted by centrifugation in a microcentrifuge at 20,000 rpm and 4 °C for 5 min, and the supernatant including the cytosolic extract was run onto 10% SDS-PAGE, then the proteins were transferred to a nitrocellulose membrane  using Towbin transfer Buffer. After incubating membranes in blocking buffer (5% low-fat milk in 100 mM Tris-HCl (pH 7.5), NaCl (145 mM), and 0.05% Tween 20; TBST), primary antibodies were added, and membranes were incubated overnight at 4 °C with gentle rocking. Membranes were washed with the buffer solution and incubated with secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. Immunoreactivity was visualized by ECL. Protein expressions of TLR3, TLR4, NF-κB and TRAF-6 were quantified by densitometry using the Scion Image Beta 4.02 software and are shown as density relative to β-actin as described by Lou et al. .x ± SD. The statistical significance between groups was calculated by one-way ANOVA followed by the Bonferroni post hoc tests for multiple comparisons. Differences were considered statistically significant at p < 0.05.All data were expressed as 25–35 in NG108-15 cells.In conclusion, the results demonstrated that ginsenoside Rg1 has a certain anti-AD activity whose possible mechanism may be related to suppressing the signaling transduction pathway of TLR3 and TLR4, and decreasing the inflammation factors induced by Aβ


Due to the fact that no drug has been effective, the search for new drugs and combinations are a priority in the HIV cure. Treatments based on nanotechnology have emerged as an innovative and promising alternative to current and conventional therapies. In this respect, nanotechnology opens up a new door for eliminating latent HIV infection. We studied the role of G1-S4, G2-S16 and G3-S16 polyanionic carbosilane dendrimers in the context of latent HIV-1 persistence. Moreover, we study the efficiency of these dendrimers in combination with latency reversal agents (LRAs) against HIV-1 infection.The major obstacle impeding human immunodeficiency virus-1 (HIV-1) eradication in antiretroviral treatment (ART) treated HIV-1 subjects is the establishment of long-lived latently infected resting CD4J89GFP lymphocyte and THP89GFP monocyte derived cell lines latently infected with HIV-1 p89GFP were used as an in vitro model of latency for our study. Viability assays by 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) were performed to determine the working concentrations of dendrimers and LRAs. Both cell lines were treated with G1-S4, G2-S16 and G3-S16 either alone or in combination with bryostatin (BRY), romidepsin (RMD) or panobinostat (PNB) for 24 and 48 h. The expression pattern of GFP was measured by flow cytometry and referred as measure of viral reactivation.The combination treatment of the dendrimers with the protein kinase C (PKC) agonist did not modify the antilatency activity in J89GFP lymphocyte cell line. Interestingly enough, G3-S16 dendrimer alone and its combination with BRY, RMD or PNB showed a significant increased expression of GFP in the THP89GFP monocyte cell line.We showed for the first time that nanoparticles, in this case, G3-S16 anionic carbosilan dendrimer may play an important role in new treatments against HIV-1 infection. These latently-infected cells are a permanent source for virus reactivation and lead to a rebound of the viral load after interruption of cARTs [Human immunodeficiency virus-1 infection can be treated effectively in the developed world, using new combinations of antiretroviral treatments (cARTs). Despite prolonged cARTs, the persistence of HIV-1 in resting CD4of cARTs .Various therapeutic interventions to eradicate HIV-1 focus on the stimulation of HIV-1 production from latently infected cells. These interventions involve the use of latency-reversing agents (LRAs) such as prostratin (PST), bryostatin-1,2 (BRY), panobinostat (PNB), and romidepsin (RMD) –9. LRAs There are two major problems in HIV-1 infection: (1) virus persistence in reservoirs in a latent form integrated into the host genome, and virus replication when cells undergo activation; (2) inhibition of current ARV drugs with retrotranscription or with HIV-1 protease. This strategy presents additional troublesome such as the appearance of resistance. Drugs that interfere with ARV entry such as enfurtivide, which inhibits viral and cell membrane fusion, have been postulated as a possible alternative. However, the first cases of resistance have already been described .In the last decade, the nanotechnology has been improved by the development and discovery of a wide range of novel nanoparticles. These new nanotechnology applications are easy to design, develop and synthesize. Dendrimers are promising nanoparticles, described as highly branched tree-like molecules between 1 and 40 nm . We workOur objective is to study the potential use of our polyanionic carbosilane dendrimers that will be applied in the “shock and kill” therapy increasing the HIV-1 reactivation and avoiding new HIV-1 infections.Polyanionic carbosilane dendrimers G1-S4 with 4 sulfate groups in periphery, G2-S16 with 16 sulfonate groups in the periphery, and G3-S16 with 16 sulfate groups in the periphery were synthesized and analyzed according to methods reported by the Dendrimers for Biomedical Applications Group of University of Alcalá  , 16. StoThese measurements were done in a Zetasizer Nano ZS  at 25 °C using disposable Malvern plastic cuvettes (1 ml), by solving 1 mg of dendrimers G1-S4, G2-S16 and G3-S16 in purified water, which was previously filtered through 0.22 µm filter.J89GFP lymphocyte and THP89GFP monocyte cell lines , are derived cell lines latently infected with recombinant HIV-1 p89 GFP. J89GFP cell line was maintained in RPMI complemented with 5% FBS, 125 mg/ml ampicillin, 125 mg/ml cloxacillin and 40 mg/ml gentamicin . THP89GFP cell line was maintained in ultra-low attachment culture plates in DMEM complemented with 5% FBS, 125 mg/ml ampicillin, 125 mg/ml cloxacillin and 40 mg/ml gentamicin  and were maintained according to the protocol described by Kutsch et al. .Bryostatin-1,2 (BRY) and prostratin (PST) were obtained from Sigma-Aldrich , panobinostat (PNB) and romidepsin (RMD) were obtained from Selleck Chemicals . Drugs were dissolved in dimethyl sulfoxide (DMSO)  to prepare stock solutions. DMSO concentration in cell cultures was lower than 0.001%.4 of J89GFP or 2.5 × 104 THP89GFP cells were seeded in 96-well plates and treated with the desired compound for 24 or 48 h. After incubation period, culture medium was discarded and 220 μl of a 1:11 MTT/OptiMEM solution was added to the cultured cells. After 3 h, the supernatant was removed, and formazan crystals were dissolved in 200 μl DMSO . All points were performed in triplicate. DMSO 10% was used as death cellular control. The concentration range of each compound examined in this study is in agreement with previously published results [The mitochondrial toxicity of compound concentrations was tested by the 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay  according to manufacturer’s instructions in G1-S4, G2-S16 and G3-S16 dendrimers for 24 and 48 h and BRY, PST, PNB or RMD for 24 h. Briefly, 6 × 10 results , 19.® Non-Radioactive Cytotoxicity  following manufacturer’s instructions. Briefly, 3 × 104 cells were seeded in 96-well plates and treated with the desired compounds and their combinations for 24 h and 48 h. After incubation period, cells were lysed in 0.9% Triton X-100  for 45 min at 37 °C. After incubation, 50 μl of LDH reagent  was added for 30 min at room temperature, protected from light absorbance was read in a Berthold Plate Reader at 490 nm. All points were performed by triplicate.Cell membrane integrity was measured by the lactate deshidrogenase (LDH) assay CytoTox 965 in 24-well culture plates and ultra-low attachment plates, respectively. Subsequently, J89GFP and THP89GFP were treated with BRY, G2-S16 or G3-S16 for 48 h at 37 °C. After incubation, both cell lines were treated with NUCLEAR-ID® Blue/Red cell viability reagent  following manufacturer’s instructions and visualized in a Leica TSC SPE confocal microscope. All points were performed in duplicate. DMSO 10% was used as death cellular control.GFP expression and LIVE/DEAD cells were analysed by confocal microscopy. J89GFP and THP89GFP were seeded at a density of 3 × 105 in 24-well culture plates and ultra-low attachment 24-well culture plates respectively, and after were stimulated with the indicated compounds. At least 30,000 cells were analysed by flow cytometry. The integrated mean fluorescence intensity  of live cells was used as a measure of HIV-1 reactivation.GFP-fluorescence pattern measured by flow cytometry was used to determine viral reactivation in J89GFP and THP89GFP cell lines. J89GFP and THP89GFP cells were seeded at a density of 4 × 10p < 0.05; **p < 0.005; ***p < 0.001).Statistical analysis was performed using GraphPad software Prism v.5.0  between two groups  were assessed by using a paired t-test.  or G3-S16 (up to 1 µM) did not show toxicity following 24 and 48 h of treatment. However, G1-S4 reduced J89GFP cells viability measured by MTT at 48 h.Potential z measurements were performed to study the aggregation ratio in aqueous solution. Due to technical issues with the small size of G1-S4, potential z for this dendrimer was not available. On the other hand, G2-S16 dendrimer z potential was − 74.0 mV showing high long-term stability in aqueous solutions which demonstrate a good biocompatibility in cell culture. G3-S16 showed a higher z potential, − 46.2 mV. These results agree with the viability data obtained by MTT and LDH assays, indicating that just 1 µM was the maximum non-toxic concentration for G3-S16, while for G2-S16 was ten times higher, 10 µM.Reactivation was measured by determining GFP expression in living cells. Confocal microscope images of non-treated, G2-S16, G3-S16, BRY and DMSO 10% were taken to confirm the expression of GFP in live cells Fig. . ResultsOur dendrimers are directed for a possible therapeutic treatment. In this context we have previously shown that these dendrimers inhibit HIV-1 infection and can be used in combination with different antiretrovirals –27. HoweThe HIV-1 reactivation effect of BRY, PST, PNB and RMD were analysed as individual drugs or in combination with G1-S4, G2-S16 or G3-S16 dendrimers at various ratios. The concentrations of the dendrimers were based on the non-toxic concentration 10 µM G1-S4, 10 µM G2-S16 and 1 µM G3-S16 previously selected. The selection of LRA concentrations were performed, taking into account the maximum non-toxic concentration of each LRA based on the scientific literature, 100 nM BRY, 20 µM PST, 40 nM PNB, and 20 nM RMD. After 24 h and 48 h of exposure, the reactivation effect was measured by flow cytometry and expressed as GFP (integrated MFI or iMFI) (Fig. Our results indicate the enhanced GFP expression with the treatment of the LRAs alone in J89GFP cell line at 24 h of exposure. The PST induced the highest response at 55% of EGFP expression. In the case of the dendrimers alone, the GFP expression was not modified in regards of the non-treated control. The combination treatment of LRAs and dendrimers indicate that the antilatency activity of the LRAs was not modified, even in some cases it tends to increase slightly in combination with G1-S4, G2-S16 or G3-S16 dendrimers. At 48 h BRY, PST, RMD or PNB tend to increase the GFP expression in J89GFP cell line. Nevertheless, RMD in combination with G1-S4 showed an increased tendency in the expression of GFP. Neither of the three dendrimers studied alone show any variation in the GFP expression in the lymphocytic derived cell line. The combination of either G1-S4, G2-S16 or G3-S16 with BRY and PST in J89GFP lymphocytic cell line at 48 h did not modify the GFP expression. Our data indicate that the combination treatment of our dendrimers with the PKC agonist did not modify the antilatency activity.In the THP89GFP, monocytic derived cell line, we obtained different results. The GFP expression in the single LRAs treatment at 24 h was greatly increased in comparison with the J89GFP lymphocytic cell line, reaching 80% of GFP expression with BRY. The LRAs combination with G1-S4 or G2-S16 dendrimer did not show any variation of the GFP expression in regards of treatment of LRAs alone. Surprisingly, in THP89GFP monocytic cell line, the G3-S16 dendrimer alone treatment showed a significant increased expression of GFP reaching 50%. We hypothesize that high size of G3-S16 in addition to sulfate groups in its periphery activate the monocytes, thus unleashing transcription factors such as NF-Kb or Sp1, forcing the viral reactivation and transcription . In the


Bacterial diversity was analyzed by denaturing gradient gel electrophoresis (DGGE). Total bacteria and cellulolytic bacteria abundance was detected by quantitative PCR. Results showed that AHA addition significantly decreased the rate of ammonia formation when urea was supplemented. Urea and AHA supplementation significantly increased the bacterial community diversity level according to the Shannon–Weiner index of 16S DGGE images. Furthermore, ruminal bacterial profiles were separated by ammonia release rate when urea was supplemented, according to the DGGE and hierarchical cluster analysis. Urea supplementation reduced the abundance of cellulolytic bacteria, such as Ruminococcus albus, R. flavefaciens, Fibrobacter succinogenes, and Butyrivibrio fibrosolvens, but inhibition of urea hydrolysis by AHA addition alleviated the reductions during the early period of incubation. In conclusion, slow release of ammonia induced by urease inhibitor influenced the ruminal bacterial diversity level and lessened the inhibition of total bacteria growth at the incubation of 12 h and F. succinogenes during the early period of incubation.The objective of this experiment was to evaluate the effects of urea hydrolysis rate on ruminal bacterial diversity level and cellulolytic bacteria abundance in vitro. To control urea hydrolysis rate, urea and urease inhibitor  were supplemented to a 2 × 2 factorial design, with urea supplemented at 0 or 20 g/kg dry matter (DM) of substrate, and AHA equivalent to 0 or 450 mg/kg DM of substrate. Ruminal fluid was collected from three Chinese Holstein dairy cows, fed a TMR, and incubated at 39 °C for 12 h after the addition of urea and AHA. Rumen fermentation parameters, which indicated the rate of ammonia formation (including ammonia-nitrogen (NH During the early and middle lactation periods in dairy cows, high dietary energy, and crude protein (CP) are required to maintain high milk production. Urea, a kind of non-protein nitrogen, is usually supplemented to the diets of dairy cows, in order to compose microbial crude protein (MCP), and reduce the cost of feeding . HoweverVarious strategies are implemented to reduce the rapid hydrolysis of dietary urea into ammonia in the rumen, such as the application of urease inhibitor to diet . It is rRuminococcus albus, R. flavefaciens, and Fibrobacter succinogenes, synthesize enzymes  that can incorporate ammonia into carbon skeleton to form different amino acids for protein synthesis and bacterial growth .The experimental design was a 2 × 2 factorial arrangement with urea equivalent to 0 and 20 g/kg of substrate , and with acetohydroxamic acid (AHA) at 0 and 450 mg/kg DM. The AHA was purchased from a commercial company . The maximal recommended dose was used. To name the treatments, U and UI were represented of urea and urease inhibitor, respectively. Thus, the four treatments were U0UI0, U0UI450, U2UI0, and U2UI450. The experiment was separately run three times on different days of collecting mixed rumen fluids, and each treatment was applied in triplicate at each experimental day.2. The buffer solution was described by 2 · 2H2O, 0.0100 g FeCl3 · 6H2O, 0.0125 g MnCl2 · 4H2O, 0.0074 g ZnCl2 · H2O, 0.0013 g CoCl2 · 6H2O, and 0.0016 g CuCl2 · 2H2O, 250 ml of buffer which contained 9.75 g NaHCO3, 250 ml of mineral solution which contained 3.59 g Na2HPO4 · 12H2O, 1.55 g KH2PO4, 0.15 g MgSO4 · 7H2O, 1.25 ml of resazurin 0.1% (w/v), and two ml of reducing solution which contained 0.5 g Na2S · 9H2O. The buffered ruminal fluid (75 ml) with initial pH 6.8 was anaerobically dispensed into a serum bottle (100 ml) containing 1.0 g ground basic diet with the appropriate dosages of urea or AHA or both. The basic diet was formulated the same as the total mixed ration for ruminal fluid donors, and contained (per kg): 177 g alfalfa hay, 175 g corn silage, 51 g oaten hay, 56 g cotton feed, 37 g apple pulp, 67 g sugar beet pulp, 27 g molasses (30%), 181 g steam-flaked corn, 56 g soybean skin, 65 g soybean meal, 39 g extruded soybean, 25 g dried distiller’s grains, 26 g double-low rapeseed meal, 2.75 g Ca(HCO3)2, 2.75 g CaCO3, 3.5 g NaCl, and 6.0 g NaHCO3, with the following chemical composition (per kg DM): 165 g CP, 355 g neutral detergent fiber, 217 g acid detergent fiber, and 65 g ether extract. There were 72 bottles (four treatments × three replicates × six sampling time points) used in total. Bottles were purged with oxygen free CO2 before being sealed with butyl rubber stoppers plus crimped aluminum seals. Incubation was conducted at 39 °C for 12 h in an incubator with intermittent manual exhaust by inserting syringe needle.On each experimental day, ruminal fluid was collected from three ruminally cannulated Chinese Holstein dairy cows approximately 4 h after feeding a total mixed ration. Ruminal fluid from the three cows was combined and rapidly filtered through four layers of sterile cheesecloth into an Erlenmeyer flask containing buffer solution  at 39 °C under a continuous flow of CO3-N) concentration was determined using the sodium salicylic acid and hypochlorous acid spectrophotometric method  and oxygen-free saline solution twice, and the eluent was added to the liquid phase. The liquid phase was centrifuged at 500×g for 15 min under 4 °C to remove feed particles and protozoa, and the supernatant was centrifuged at 22,000×g for 20 min under 4 °C to obtain ruminal bacteria. Ruminal MCP synthesis was estimated by the ratio of purines to isolated bacterial nitrogen  on ice in each sampling time point. Fresh culture fluids were dispensed immediately into different centrifuge tubes and frozen at −20 or −80 °C for further analysis. Ammonia-nitrogen  using the following program: initial denaturation for 3 min at 94 °C; 10 cycles of 94 °C for 30 s, 60 °C for 30 s, decreasing 0.5 °C every cycle, and 72 °C for 30 s; 20 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s; and finally, holding at 72 °C for 5 min. The PCR products were confirmed by electrophoresis on a 1.5% (w/v) agarose gel stained with 5% GoldView solution. Before denaturing gradient gel electrophoresis (DGGE) analysis, the PCR products of samples from three separate days for the same treatment were mixed together as one sample, so finally there were 8 PCR products ) subjected to DGGE analysis using the DCode Universal Mutation Detection System . Gradient gels containing 8% (w/v) polyacrylamide (37.5:1 acrylamide/bis-acrylamide) were prepared with 40–60% urea formamide denaturant. The 20–25 μl each mixed PCR products sample with DNA of 200 ng was used for gel loading. The electrophoresis was run at 85 V for 16 h at 60 °C. The DNA bands in the gels were visualized by silver staining according to the procedures described by Primers used to amplify the V3 region of the 16S rRNA gene were GC-338F: (GC clamp-ACTCCTACGGGAGGCAGCAG) and 533R (TTACCGCGGCTGCTGGCAC) . The PCR2O. The qPCR program consisted of the following: 95 °C for 30 s; 40 cycles of 95 °C for 5 s, 60 °C for 34 s (with fluorescence collection). Both standard and DNA sample were routinely run in triplicates using the same master mix in the same PCR plate. A negative blank (without DNA template) was also run for each primer pair. The copies of total bacteria and ruminal cellulolytic bacteria were determined by relating their Cq values to their standard curves, respectively. Only the data of qPCR efficiencies between 90% and 105% were used for analysis  in an ABI 7500 Real Time PCR System . The primers are shown in analysis . Data weP < 0.05.All the statistical analyses were conducted using the PROC MIXED procedure of SAS 9.2 . The data on populations of total bacteria and ruminal cellulolytic bacteria were converted to a logarithm base before statistical analysis. Data from urea hydrolysis, Shannon–Weiner index and bacterial populations were analyzed with urea, AHA, incubation time, and urea × AHA interaction included in the model as fixed effects. Data from separate days were used as repeated measures. Orthogonal polynomial contrasts were performed to compare the treatments. All data were presented as least squares means, and significant differences were declared at 3-N was increased by urea supplementation through the whole incubation (P < 0.05), but it was numerically decreased by AHA addition within 2 h of incubation (significantly in 1 and 2 h). Urease activity was stimulated by urea supplementation within 6 h of incubation , but it was numerically inhibited by AHA addition within 2 h of incubation (significantly in 2 h). Additionally, urease activity of U2UI450 treatment was inhibited by 51.7% at 1 h after incubation, and by 35.7% at 2 h after incubation, compared to that in U2UI0 treatment. After incubation of 12 h, there was no statistical differences on MCP concentration among treatments.The urea-N concentration could not be detected in the treatment with urea supplementation only by 1 h postincubation, but it gradually decreased until 6 h of incubation in the treatment with both urea and AHA supplementation . The convs. U2UI450). The ruminal bacterial profiles were separated by ammonia release rate when urea was supplemented, according to the DGGE and hierarchical cluster analysis. The Shannon–Weiner index showed that treatments with urea supplementation had lower (P < 0.05) diversity of predominant species, while AHA addition (U2UI450) could increase (P < 0.05) the bacterial diversity.To verify the observed diversity variation among treatments at different incubation stages, the hierarchical cluster analysis was performed using the pooled samples . TreatmeP < 0.01). After incubation of 12 h, total bacteria population of U2UI0 treatment was lower than other treatments (P < 0.05) (R. flavefaciens (P = 0.03) and Butyrivibrio fibrosolvens (P < 0.01) at 0.5 h of incubation, and populations of these two bacteria in treatment of U2UI0 were lower than their populations in treatment of U2UI450 at 0.5, 4, and 12 h of incubation. For F. succinogenes, urea supplementation decreased its population at 0.5 h of incubation (P = 0.05), but AHA addition increased (P = 0.03) it. The F. succinogenes population of U2UI0 treatment was lower than other treatments (P < 0.05) at 0.5 h of incubation.Interactive effects between urea and AHA supplementation were noticed on total bacterial population within 4 h of incubation ( < 0.05) . InteracRumen simulation systems have been used in the evaluation of rumen fermentation manipulation and feeds nutrients degradation in order to avoid the use of animals or decrease study costs . In thisUrea accompanied with urease inhibitor is usually used as a dietary supplement to provide degradable nitrogen, and acts as a solution for the rapid hydrolysis of urea in rumen. In this study, urea hydrolysis and ammonia formation were successfully inhibited by the interaction of urease inhibitor (AHA) and urea supplementation, which was in agreement with previous in vivo and in vitro studies . Brent, 3-N concentration required to support maximum synthesis of microbial protein was 12.8 mg/100 ml in lactating Holstein cows fed a corn silage-based diet with graded levels of urea  using in vitro fermentation technique  released from 0.02 g of dietary urea hydrolysis altered the level of nitrogen in cultural solutions.The rumen microbial community inhabiting in rumen is characterized by high population density, broad diversity and complex interactions . It was 3-N to synthesize microbial protein in the rumen  addition significantly decreased the rate of ammonia formation when urea was supplemented. Slow release of ammonia induced by AHA influenced the ruminal bacterial diversity level (depend on the result from DGGE cluster) and lessened the inhibition of total bacteria growth at the incubation of 12 h and 10.7717/peerj.5475/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.5475/supp-2Supplemental Information 2Click here for additional data file.