These superstructures evidenced by voltage plateaus in the electrochemical curves limit the Na+ ion transport kinetics and cycle performance in rechargeable batteries. Here we show that such Na+/vacancy ordering can be avoided by choosing the transition metal ions with similar ionic radii and different redox potentials, for example, Cr3+ and Ti4+. The designed P2-Na0.6[Cr0.6Ti0.4]O2 is completely Na+/vacancy-disordered at any sodium content and displays excellent rate capability and long cycle life. A symmetric sodium-ion battery using the same P2-Na0.6[Cr0.6Ti0.4]O2 electrode delivers 75% of the initial capacity at 12C rate. Our contribution demonstrates that the approach of preventing Na+/vacancy ordering by breaking charge ordering in the transition metal layer opens a simple way to design disordered electrode materials with high power density and long cycle life.Most P2-type layered oxides exhibit Na Sodium-containing layered oxides are promising battery cathodes, but their performance suffers from the formation of sodium ion-vacancy ordered superstructures. Here, the authors present a P2-Na0.6[Cr0.6Ti0.4]O2 layered oxide with disordered cations, leading to high battery performance. Such ordered superstructures have distinct voltage plateaus in electrochemical curves+ ion diffusion coefficient (by up to two orders of magnitude6) layers, for example, NaxMnO2  we show that P2-Na0.6[Cr0.6Ti0.4]O2 synthesized through a simple solid-state reaction is Na+/vacancy-disordered at any sodium content in the studied range of x=0.33–1.0 in Nax[Cr0.6Ti0.4]O2 and temperature from 3 to 1,073 K that leads to its very high ionic conductivity and ability to withstand high charge/discharge rates.Here we intentionally chose the transition metal ions with very similar ionic radii and substantially different redox potential versus Na, such as CroO2 ref.  and NaxVVO2 ref.  that in 2CO3, Cr2O3 and TiO2. The inductively coupled plasma (ICP) result confirms the composition of Na0.605[Cr0.6Ti0.4]O2. The morphology of the sample was investigated with scanning electron microscopy (SEM). As shown in the inset of 0.6[Cr0.6Ti0.4]O2, two questions of crucial importance were whether Cr and Ti are ordered in the octahedral layers and whether Na atoms are ordered over Na1 and Na2 prisms. Both aspects were studied in detail using NPD, which was clearly a technique of choice in this case. First, in contrast to X-ray diffraction, NPD is extremely sensitive to Cr and Ti distribution because of an opposite sign of the neutron-scattering lengths . Second, again in contrast to X-ray diffraction, in the case of random distribution of Cr and Ti the contribution of transition metals to the total diffraction pattern is rather small (bcoh(Cr0.6Ti0.4)=0.833 fm) that makes NPD more sensitive to the contribution from the sodium sublattice (bcoh(Na0.6)=2.18 fm).This oxide was synthesized by a simple solid-state reaction using precursors of Na3/mmc no. 194) with a unit cell  and random distribution of Cr/Ti and Na over a single octahedral site and two trigonal prismatic sites, respectively, was successfully used to analyse the data  sites that significantly improved refinement quality. The Rp and Rwp reduced from 2.30% and 2.99% to 2.08% and 2.73%, respectively, with only two additional variables, which is certainly statistically significant4041xO2 compositions with M1=V–Ni and M2=Ti, Li, Mg, and so on because of the asymmetric local coordination of Na by M1 and M2. Uncompensated electrostatic repulsion displaces Na+ from prism centres.At room temperature the high-quality NPD data unambiguously revealed absence of supercell diffraction peaks and therefore a high symmetry model /diethyl carbonate (DEC) as the electrolyte is shown in 0.6[Cr0.6Ti0.4]O2 electrodes in the voltage ranges of 2.5–3.85 and 0.5–2.5 V, respectively. It can be seen that all the charge and discharge curves show smooth slopes without plateaus, indicating the sodium deintercalation and intercalation through a single-phase reaction. This is further supported by the in situ XRD results as discussed later. This behaviour is very different from some other P2-type electrodes such as NaxCoO2, NaxVO2, where multiple plateaus were observed during sodium deintercalation/intercalation, which is related to the formation of Na+/vacancy and charge-ordered superstructures90.6[Cr0.6Ti0.4]O2, the disordered distribution of Cr and Ti in the transition metal layer as discussed above is responsible for such sodium storage mechanism.The sodium storage performance of P2-Naca. 3.5 V, which is much higher than that of O3-type NaCrO2 (ca. 3 V) with the same Cr3+/Cr4+ redox couple0.6[Cr0.6Ti0.4]O2 and NaCrO2 samples. The reversible discharge capacity is ∼74 mAh g−1, corresponding to 0.26 Na deintercalation/intercalation per formula unit. The rate capability of the Na0.6[Cr0.6Ti0.4]O2 electrode was also tested in a sodium-ion battery. It can be seen from −1 at constant current rates of C/10, C/5, C/2, 1C and 2C, respectively. The capacity retention at 1C is 82% of the initial capacity. The long-term cyclic stability of this material is also excellent at current rates of 0.1C and 1C as shown in 0.6[Cr0.6Ti0.4]O2 electrode exhibits over 200 cycles with capacity retention of 94% at a current rate of 1C. The Coulombic efficiency after initial cycles reaches nearly 99.4%.Interestingly, the average Na storage voltage for the positive electrode is ca. 0.8 V, which is similar to P2-Na0.66[Li0.22Ti0.78]O2  layer on the surface of the electrode. It is noted that when a NaClO4-based electrolyte was used, an even much lower Coulombic efficiency of 54% was observed as shown in 6-based electrolyte is much thinner than that from the NaClO4-based electrolyte (4-based electrolyte (x=0.6–1) in the Nax[Cr0.6Ti0.4]O2 negative electrode, resulting in slower Na+ ion diffusion because of the strong repulsive electrostatic interactions among Na+ ions. Again, it can be seen from 0.6[Cr0.6Ti0.4]O2. This performance is much better than that of other P2-type oxides with ordered structure69131516171820For the negative electrode side, the average sodium storage voltage is a0.66Li0.Ti0.78O2 ctrolyte , which cctrolyte . In term0.6[Cr0.6Ti0.4]O2 as both negative and positive electrodes. The greatest advantage of using the same materials as both negative and positive electrodes is to significantly decrease the material's processing cost. Preliminary results show that the full cell delivers an average operating voltage plateau at ∼2.53 V and extraordinary rate  and X-ray absorption spectroscopy (XAS) were performed. 2,3 and Ti-L2,3 edges for the pristine Na0.6[Cr0.6Ti0.4]O2 sample and the Na0.6[Cr0.6Ti0.4]O2 electrodes charged to 3.85 V and discharged to 0.5 V, respectively. In the case of positive electrode, the Cr-L2,3 edge shifts to a higher energy while Ti-L2,3 edge keeps unchanged on charged to 3.85 V. The shift of Cr-L2,3 could be assigned to the formation of Cr4+, which is further supported by the XAS results as shown in 2,3 edge changes significantly, corresponding to the Ti4+/Ti3+ reduction during Na insertion3+/Cr4+ is responsible for the charge compensation on Na extraction, whereas Ti4+/Ti3+ is involved in the charge compensation during Na insertion, which is in excellent agreement with our initial design.To elucidate the structure evolution during sodium deintercalation and intercalation, we carried out the electrochemical + ordering at any temperature and sodium content such as known to occur in NaxCoO2 0.6[Cr0.6Ti0.4]O2 demonstrates normal Debye-type thermal expansion without anomalies  simulations as implemented in the VASP (Viena ab initio simulation package) code+/vacancy and Cr/Ti using a 108-atom supercell through the ATAT code0.6[Cr0.6Ti0.4]O2 system. The mean square displacements (MSDs) of Na+ ion for P2-Na0.6[Cr0.6Ti0.4]O2 at various temperatures are displayed in + ion migration can be observed for all these temperatures. As the temperature increases, the Na+ ions diffuse more and more easily. To obtain the diffusion coefficient (D), the slope of each curve is fitted in the range from 2 to 8 ps. Ea) is extracted from the slope of the Arrhenius plot. The calculated activation energy barrier for P2-Na0.6[Cr0.6Ti0.4]O2 is (0.35±0.07) eV, which is comparable to the experimental measurements, 0.24 eV, indicating the effectiveness of our simulations. The detailed diffusion process of Na+ ions is shown in + ions are given to illustrate the migration pathways. The positions of Cr, Ti and O atoms are fixed at their initial ones for clarity, since no breaking of Cr–O and Ti–O bonds is found in all the simulations. It is clear to see that the Na+ ions migrate within two-dimensional channels, since no diffusion between the Na+ layer and the transition metal layer can be found. The top view of trajectories in each Na+ layer reveals similar pictures with the section of nuclear density given in To obtain a microscopic picture of the Na3+ and Ti4+ with very similar ionic radii and a substantial difference in redox potential, we designed a P2-Na0.6[Cr0.6Ti0.4]O2-layered oxide simultaneously Cr/Ti and Na+/vacancy-disordered in the complete studied wide range of sodium content and temperature. It was demonstrated that this cation-disordered material can function as both positive and negative electrodes with average operation voltages of 3.5 and 0.8 V, corresponding to the redox couples of Cr3+/Cr4+ and Ti3+/Ti4+, respectively. A symmetric sodium-ion battery using the same P2-Na0.6[Cr0.6Ti0.4]O2 electrode material demonstrates charge/discharge curves free of voltage plateaus, outstanding rate performance and long cycle life. Our study highlights the importance of designing a disordered transition metal/charge arrangement in the layered oxides as a promising strategy to improve their Na-storage performance in terms of rate capability and long cycle stability for the development of high-power and long-life room-temperature sodium-ion batteries.In summary, through selecting transition metals of Cr2CO3 (99%), Cr2O3 (99%) and TiO2 . A phase-pure compound was obtained when an excess of 2 mol% Na2CO3 was used. The starting materials were ground in an agate mortar and pressed into pellets under pressure of 20 MPa. Then, the pellets were heated at 1,000 °C for 15 h under Ar atmosphere in an alumina crucible.The resulting material was prepared by a solid-state reaction using precursors of Naλ1=1.54060 Å, λ2=1.54439 Å) and a LynxEye_XE detector. The XRD pattern was refined using the TOPAS software based on the Rietveld method. In the in situ XRD studies, the working electrode was prepared using PVDF as binder on an Al foil. A specially designed Swagelok cell equipped with an X-ray-transparent beryllium window was used for the in situ measurements. The in situ XRD patterns were collected with an interval of 30 min for each 2θ scan from 10° to 80° on charge and discharge at a current rate of C/10, between 0.5∼2.5 and 2.5–3.85 V versus Na+/Na. An aluminium foil was placed between the cathode electrode and the beryllium window, to prevent beryllium oxidation at high operating voltages.The morphologies of the materials were investigated using a scanning electron microscope (Hitachi S-4800). Powder X-ray diffraction was performed using a Bruker D8 Advance diffractometer equipped with a Cu Kα radiation source . The slurry was stirred until the colour of the solution-fade completely to obtain the sodiated products. The products were washed by DME several times, and dried in the vacuum for overnight. Chemically sodium deintercalation of the sample was accomplished by reaction with a strong oxidant of nitronium tetrafluoroborate 2BF4 was dissolved in acetonitrile and Na0.6[Cr0.6Ti0.4]O2  was added to the resulting solution. The slurry was stirred overnight at room temperature, filtered under vacuum and then finally washed several times with acetonitrile.The Na,NPD data were collected using the high-resolution powder diffractometer Echidna at the OPAL research reactor  using wavelengths of 1.6215 Å. The NPD data analysis with Rietveld and Maximum Entropy Methods were performed using General structure analysis system52ex situ XAS data at the Cr K-edge were recorded at room temperature in the transmission mode at beam line BL14W1 of the Shanghai Synchrotron Radiation Facility (SSRF), China. The station was operated with a Si (111) double crystal monochromator. The photon energy was calibrated with the first inflection point of Cr K-edge in the relevant metal foil. Analysis of the XAS spectra was carried out by using programme code of IFEFFITEELS spectra were collected with a Gatan GIF spectrometer attached to a JEOL 2010F field emission microscope operating at 200 KeV. The transmission electron microscope was operating at a diffraction mode with a camera length of 8 mm. The spectrometer was set at 0.2 eV per channel with a collection aperture of 1 mm.The EELS spectrum background was subtracted by power law fitting. The electronic states of the Cr during the charge and discharge processes were probed using X-ray absorption near-edge structure spectroscopy techniques at Cr K-edges. The 4 or 0.8 M NaPF6 in EC/DEC (4:6 in volume). The coin-type (CR2032) cells were assembled with pure sodium foil as the counter electrode, and a glass fibre as the separator in an argon-filled glove box. The charge and discharge measurements were carried out on a Land BT2000 battery test system  in voltage ranges of 0.5–2.5 and 2.5–3.85 V under room temperature. A sodium-ion full cell was constructed using P2- Na0.6[Cr0.6Ti0.4]O2 as both the negative and positive electrodes in a 2032 coin-type cell. The weight ratio of the two electrodes (negative/positive) was 1:1.3–1.4 (ref. −1).The working electrode was prepared by spreading the slurry of the active materials (75 wt.%), acetylene black (20 wt.%) and the polyvinylidene fluoride PVDF (5 wt.%) binder on the Al foil. The working electrodes were dried at 100 °C under vacuum for 10 h. The electrolytes are 1 M NaClO1.4 ref. . The ful+/vacancy and Cr/Ti, the SQS is constructed using a 108-atom supercell through the ATAT code+ ion transport properties in P2-Na0.6[Cr0.6Ti0.4]O2. The simulations are performed using the density functional theory as implemented in the VASP code47p,3s), Cr, Ti and O orbital are treated as valence states. The energy cutoffs for plane wave basis set and augmentation charges are 500 and 756 eV, respectively. The SQS structure is first optimized to obtain the equilibrium lattice parameters and ionic positions on a 3 × 3 × 2 k-mesh. The energy and force convergence criterion for the relaxation are 10−5 eV and 0.01 eV Å−1, respectively. Then, the FPMD simulations are carried out for 10 ps at each temperature by a Nose thermostat58To simulate the disordered configurations of Nari(t) is the position of the i-th Na+ ion at the time t, and the average is over the time steps and all the Na+ ions. According to the Einstein equation, the slope of the MSD presents the diffusion coefficient D,where +-ion diffusion can be extracted from the diffusion coefficients at various temperatures according to Arrhenius equation.The activation energy barrier for Nain situ XRD measurements; Y.W. performed ex situ XAS measurements, transmission electron microscope and EELS; M.A. collected the NPD data and refined the NPD results; R.X. performed the first principles calculations, Y.-S.H., R.X. and M.A. wrote the paper; all the authors participated in analysis of the experimental data and discussions of the results as well as preparing the paper.Y.-S.H. and M.A. designed this work; Y.W. carried out the synthesis and electrochemical experiments; Y.W. performed How to cite this article: Wang, Y. et al. P2-Na0.6[Cr0.6Ti0.4]O2 cation-disordered electrode for high-rate symmetric rechargeable sodium-ion batteries. Nat. Commun. 6:6954 doi: 10.1038/ncomms7954 (2015).Supplementary Figures 1-10 and Supplementary Tables 1-3


The reference population comprised 50 DMD-untreated patients with RRMS. After 24 months, 43 of the untreated patients switched to DMDs. The four groups of patients were followed up for an additional 24 months. At 48 months the mean standard deviation number of new CLs was significantly lower in patients treated with sc IFN β-1a  compared with im IFN β-1a  and glatiramer acetate . Significant reductions in CL accumulation and new white matter and gadolinium-enhancing lesions were also observed in the 43 patients who switched to DMDs after 24 months, compared with the 24 months of no treatment. Concluding, this study confirms that DMDs significantly reduce CL development and cortical atrophy progression compared with no treatment.Cortical lesions (CLs) and atrophy are pivotal in multiple sclerosis (MS) pathology. This study determined the effect of disease modifying drugs (DMDs) on CL development and cortical atrophy progression in patients with relapsing-remitting MS (RRMS) over 48 months. Patients ( Despite the older age, the apparently milder disease evolution, and the lower cortical lesion load of the untreated group  [P < 0.001). Moreover, a significant lower decrease in gray matter fraction was observed in DMD-treated patients compared with untreated patients (P = 0.023). Here we report a 24-month extension study that analyzed the long-term effects of DMDs on cortical pathology and evolution in patients who, after 24 months of no treatment, began active treatment.The cortical pathology of multiple sclerosis (MS) is characterized by the accumulation of cortical lesions (CLs) –3 and a ication) , each reβ-1a (44 μg three times weekly), im IFN β-1a (30 μg once weekly), or sc glatiramer acetate (20 mg once daily), while 50 patients with RRMS remained untreated. This untreated population was not randomized and reasons for not treating included both physician- and patient-driven factors, such as a relatively benign clinical course (low relapse rate and low WM lesion load on repeated MRI scans), needle phobia, female patients who were pregnant or trying for pregnancy, and interruption of previous therapy attempts with DMDs owing to persistent adverse effects or poor treatment adherence , and philosophic or religious beliefs. Clinical disability was assessed every 6 months using the EDSS. MRI was performed at baseline and at months 12, 24, and 48 in all patients and in cases of relapse. In this study, a suboptimal DMD response was defined as the occurrence of a relapse determining an increase in disability of one EDSS point or the persistence of disease activity at MRI. After the first 24 months, since July 1 to December 31, 2010, a first-line therapy was initiated in 43/50 patients of the reference population. Given the low number of patients, therapy allocation was not randomized in this group but based on physician choice. Hence, 17 were treated with sc IFN β-1a, 12 with im IFN β-1a, and 14 with glatiramer acetate. These rather equally balanced patients were considered a unique group in the subsequent statistical analysis. The study was approved by the local Ethics Committee (HREC name: D. Piovan) and a written informed consent was obtained from all the patients.The core study has been described previously . BrieflyMRI procedure in the extension period was previously described . BrieflyP value <0.05 was considered statistically significant. Stata 12.1 software  was used for all analyses.The Wilcoxon test was used to compare the 24- and 48-month time-point data . The Huynh-Feldt epsilon correction method  was usedn = 50), 43 (86.0%) began treatment after the first 24 months. On the light of the results of the previous 24-month study and considering the low number of patients, treatment allocation was not randomized but mainly based on physician choice. As mentioned above, 17 patients initiated therapy with sc IFN β-1a, 12 with im IFN β-1a, and 14 with glatiramer acetate. This group was analyzed as a whole since the low number of patients in each subgroup did not allow a comparison between treatments. Four out of the seven patients that decided to remain therapy-free were lost during the follow-up; thus this group could not be used for statistical analyses.Of the 165 patients originally randomized to receive first-line DMDs, 129 (78.2%) completed the full 48 months of follow-up remaining on the originally allocated therapy, while 24 (14.5%) switched treatment during the first 24 months and 12 (7.3%) during the second 24 months of therapy . Since tβ-1a group (45 patients), compared with 2.3 ± 1.3 (range 0–6) in the im IFN β-1a group (41 patients) and 2.2 ± 1.5 (range 0–7) in the glatiramer acetate group (43 patients). The mean number of new CLs was significantly lower in the sc IFN β-1a group than for either of the other treatments . P < 0.001), white matter lesion mean was 2.1 ± 1.9 and 1.0 ± 0.2 (P < 0.001) and Gd+ lesions were 1.9 ± 1.0 and 0.5 ± 0.4 (P < 0.001), respectively. Mean annualized relapse rate was also significantly lower during treatment than pretreatment (P = 0.002). At month 48, the mean count of new white matter lesions and EDSS change were significantly different between sc IFN β-1a, im IFN β-1a, and glatiramer acetate, with more pronounced effects seen with sc IFN β-1a . The mean number of relapses did not differ between treatment groups.In the group that remained on initial treatment, the mean standard deviation (SD) number of new CLs at 48 months was 1.4 ± 1.0 (range 0–5) in the sc IFN β-1a), 37.1% (im IFN β-1a), and 37.4% (glatiramer acetate). Compared to baseline, at month 48, the mean ± SD percentage decrease in gray matter fraction was 1.8% ± 0.6% in the 43 patients who were untreated for 24 months and then began active treatment and 1.4% ± 0.5% (P = 0.04) in the 129 patients who remained on active treatment for the duration of the study. Percentage change from baseline in gray matter fraction did not differ significantly among the treatment groups.Mean baseline gray matter fraction was comparable across all the groups that retained the original therapy: 37.2% (sc IFN β-1a has a more rapid and pronounced effect on cortical pathology compared with both im IFN β-1a and glatiramer acetate.The initial 24-month study showed the effect of DMDs in reducing the development of CLs in patients with RRMS , and theβ-1a significantly slowed progression of brain and gray matter atrophy in RRMS over 2 years [Previous studies demonstrated that sc IFN  2 years , 16. Our 2 years , 9. Incr 2 years . We have 2 years , while t 2 years –22. Fina 2 years .Despite the limitations described previously , our stu


Oxidative stress-induced DNA damage resulted in increased H2AX(S139) phosphorylation , along with the degradation of the androgen receptor (AR), p53 and NKX3.1, upon treatment with conditioned medium (CM) obtained from activated macrophages or H2O2. Furthermore, the expression and stability of SIRT1 were increased by CM treatment but not by H2O2 treatment, although the level of ATM(S1981) phosphorylation was not changed compared with controls. Moreover, the deregulated antioxidant response resulted in upregulation of the pro-oxidant QSCN6 and the antioxidant GPX2 and downregulation of the antioxidant GPX3 after CM treatment. Consistently, the intracellular ROS level increased after chronic treatment, leading to a dose-dependent increase in the ability of LNCaP cells to tolerate oxidative damage. These data suggest that the inflammatory microenvironment is a major factor contributing to DNA damage and the deregulation of the oxidative stress response, which may be the underlying cause of the increased genetic heterogeneity during prostate tumor progression.The oxidative stress response is a cellular defense mechanism that protects cells from oxidative damage and cancer development. The exact molecular mechanism by which reactive oxygen species (ROS) contribute to DNA damage and increase genome instability in prostate cancer merits further investigation. Here, we aimed to determine the effects of NKX3.1 loss on antioxidant defense in response to acute and chronic inflammation in an  Oxidative stress contributes to the initiation, promotion and progression of carcinogenesis. Excessive levels of reactive oxygen species (ROS) are generated by exposure to oxidative stress and cause sustained DNA damage. DNA damage occurs during the initiation step of carcinogenesis, and most likely results in abnormal gene expression. Because ROS accumulation results in a subsequent failure of signal transduction at the promotion step, the cells undergo a loss of genomic fidelity during the progression step . NotablyTo reduce the oxidative damage caused by ROS, the antioxidant response system is activated through several constitutive and inducible detoxification mechanisms, including the expression of enzymes such as glutathione peroxidases (GPx), catalase, superoxide dismutases (SODs), peroxiredoxins (PRDXs), glutathione-S-transferases (GSTs), NADP (H) quinone oxidoreductase (NQO1), epoxide hydrolase, heme oxygenase (HO-1), UDP-glucuronosyl transferases (UGTs), and gamma-glutamylcysteine synthetase. The upregulated expression of these enzymes exhibits a distinct cellular defense to protect cells from oxidative damage and cancer development ,10.In addition to the mechanisms that regulate ROS, cell proliferation is controlled by stress-sensing molecules such as Sirtuin 1 (SIRT1), an NAD-dependent deacetylase that responds to the levels of redox pairs NAD+/NADH and NADP+/NADPH. Under oxidative conditions, SIRT1 deacetylates a number of transcription factors, including p53, NBS1 and FOXO, which subsequently contribute to cellular metabolic responses, such as cell cycle regulation and DNA damage ,11,12. TE. coli infected prostate lobes in mice has been shown to be correlated with reduced AR expression [NKX3.1 is an androgen-regulated gene that encodes a homeobox protein with a tumor suppressor function in prostate cells ,15. The pression . It was pression ,18 but apression . Concurr2O2 for 2 weeks to compare the effects of pro-inflammatory cytokines and ROS exposure.In this study, we aimed to identify the role of cytokine-induced NKX3.1 loss in the deregulation of the antioxidant defense during acute and chronic exposure to both cytokines and ROS. Therefore, the effect of antioxidant treatment on the inflammation- and/or oxidative stress-induced degradation of NKX3.1, AR and p53 was analyzed. Cultures of the prostate cancer cell line LNCaP were exposed to conditioned medium (CM) with adjusted amounts of pro-inflammatory cytokines (TNFα) for 24 h for acute treatment and for 2 weeks for chronic treatment. Cells were also chronically treated with H2. To achieve macrophage differentiation and cytokine production, cells (8×105) were seeded into 75-cm2 culture flasks 2 h prior to treatment. Next, PMA was added at a final concentration of 16 nM for 16 h, and the adherent clusters (differentiated monocytes) were maintained. The cells were washed twice before the addition of 20 ml of fresh medium, and the cells were then allowed to rest for 3 h. Then, lipopolysaccharide (LPS) was added at a final concentration of 10 ng/ml to induce cytokine secretion. The cells were incubated for an additional 24 h, and the supernatant (conditioned medium - CM) was collected and filtered (using a 0.22-μm filter) for further use. To ensure that the CM was cell-free, diluted CM was cultured in an empty flask (25 cm2) for one week and analyzed.The U937 monocyte cell line was cultured in RPMI 1640 medium including 10% FBS  at 37°C with 5% COBefore feeding the LNCaP cells with the collected CM (cell-free), the TNFα  levels were analyzed using an ELISA according to the manufacturer’s instructions. Because cytokine exposure is a major component of the inflammatory microenvironment, the times  and doses  for the courses of CM treatments were optimized as reported in our previous study . TNFα wa2. For the acute exposures, the CM  treatments were performed for 24 h; for the chronic exposures, the CM treatments continued for 2 weeks, and lower doses (50 and 100 pg/ml of TNFα) were used. TNFα concentrations were adjusted by diluting the CM using RPMI 1640 medium as described previously [2O2 for 2 weeks for comparison of the effects of cytokine exposure and oxidative stress.LNCaP cells were obtained from the American Type Culture Collection  and were propagated as recommended using RPMI 1640 medium supplemented with 10% FBS, L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C with 5% COeviously . A chronNKX3.1 open reading frame was amplified (using the primers F: GGATCCATGCTCAGGGTTCCGGAGCCG and R: GAATTCGGTTGTCACCTGAGCTGGCATTA) and cloned into the pcDNA4/HisMax-TOPO vector  according to the manufacturer’s instructions to obtain HM-NKX3.1 and the HM-vector constructs. Then, transfections were performed using the Fugene HD reagent  for 24 h. The cells were incubated for an additional 18 to 42 h, as appropriate.The 5 cells were seeded into 6-cm plates, and the medium was changed (w/o antibiotics). A transfection mix was prepared by adding 6 μl of Dharmafect II (tube 1) and 200 pmol of siNKX3.1, siAR or scrambled siRNA (tube 2) into 94 μl of transfection medium (w/o antibiotics and serum). After incubation for 5 min at RT, the tubes were mixed and incubated for 15 min at RT and then added onto the cells dropwise. The transfected cells were incubated for an additional 24 h before harvesting.The siNKX3.1 and siAR transfections were performed as recommended by the supplier (Dharmacon). Briefly, 4×10(S139) and pATM(S1981) , GAPDH , β-actin and SIRT1 , Caspase-3 , and β-tubulin . The NKX3.1 custom antibody was a gift from Prof. Dr. F. Saatcioglu (University of Oslo). The HRP-conjugated anti-mouse and anti-rabbit  and the AlexaFluor 488- and 594-conjugated secondary antibodies  were purchased and used as recommended by the manufacturers.The following antibodies were purchased from the manufacturers: AR , p53, pH2AX3) were seeded into 96-well plates, and the transfections were carried out on the following day. Two days later, the cells were incubated with DCFH-DA  for 30 min at 37°C. Next, the treatments were performed following gentle washes using phenol red-free medium. Finally, the fluorescence intensity was measured every 20 min for up to 3 h using a Fluoroscan fluorometer .LNCaP cells , 1% Triton X-100, 0.1% SDS, 0.1% Na deoxycholate, 1 mM EDTA, 1 mM EGTA, 140 mM NaCl) containing protease and phosphatase inhibitors. Then, the concentrations were determined using the BCA assay . SDS-PAGE and western blots were performed under standard conditions with 50 μg of protein lysate per lane. The proteins were separated on 10-12% gels and transferred to PVDF membranes  using a wet transfer blotter. The PVDF membrane was blocked with 5% dry milk in TBS-T . The primary and secondary antibody incubations were performed in TBS-T containing 0.5% dry milk or 5% BSA at RT for 1 h or at 4°C o/n. The membranes were developed using the ECL prime reagent  for 5 min and were photographed using Kodak X-Ray films in a dark room.3) were transfected with an HM vector and HM-NKX3.1 (24 h), seeded into 96-well plates  and cultured for 24 h. The treatments were performed as described, and the proliferation rate and morphological changes were monitored. Impedance values were collected every 10 min for 48 h.The Xcelligence proliferation assay platform was used for real-time measurements. Briefly, the LNCaP cells , and the yield was calculated using absorbance readings at 260/280 nm. Then, cDNA synthesis was performed using a cDNA synthesis kit  as recommended by the manufacturer.To study the expression of specific genes, quantitative RT-PCR was performed using a SYBR Green PCR kit and the LC480 PCR system . The relative abundance of each transcript was calculated using the comparative cycle threshold (CT) method with GAPDH as an invariant control. The following primers were used: GPX2_F: CAGTCTCAAGTATGTCCGT, GPX2_R: AGGCTCAATGTTGATGGT; GPX3_F: CTTGCACCATTCGGTCT, GPX3_R: CGGACATACTTGAGGGTAG; PRDX6_F: TAGTGTGATGGTCCTTCCAAC, PRDX6_R: AGCGGAGGTATTTCTTGC; QSCN6_F: GAGGCTACGTGCACTACT, QSCN6_R: CTGCAAGGCGAGCATTGA; ENOX2_F: CTGAACGTGAAGCACTG, ENOX2_R: ATCAAGACGGTGCAAGTAG; SOD1_F: TGTACCAGTGCAGGTCC, SOD1_R: GCCAATGATGCAATGGTC; SOD2_F: TGTCCAAGGCTCAGGTT, SOD2_R: CTGAAGGTAGTAAGCGTGC; NKX3.1_F: TCTATCAGCATCTGACAGGTGAA, NKX3.1_R: AGCAGGGTTTGTTATGCATGTAG; SIRT1_F: TGCGGGAATCCAAAGGATAATTCAGTGTC, SIRT1_R: CTTCATCTTTGTCATACTTCATGGCTCTATG; and GAPDH_F: CATTGCCCTCAACGACCACTTT, GAPDH_R: GGTGGTCCAGGGGTCTTACTCC.t test was applied to determine the statistical significance between pairs where necessary.Student’s 2O2 with or without the antioxidant, N-Acetyl-L-cysteine (LNAC). The CM treatments resulted in the degradation of AR, p53 and NKX3.1 in a dose-dependent manner, which is consistent with our previous studies [2O2 were almost completely restored back to basal levels by LNAC  treatments were performed for 3, 6 and 24 h. The results indicate that NKX3.1 loss was correlated with antioxidant responsive gene expression in LNCAP cells. We found that the expression of the pro-oxidants Quiescin Q6 (QSCN6) and Ecto-NOX disulfide thiol exchanger 2 (ENOX2) was increased by 2.7 and 1.3-fold relative to the controls, respectively. The antioxidant glutathione peroxidase-2 (GPX2) was upregulated 6.2-fold, and glutathione peroxidase-3 (GPX3) and peroxiredoxin-6 (PRDX6) were downregulated 12.5- and 2.4-fold, respectively. Further, the antioxidant superoxide dismutase-1 (SOD1) was downregulated 1.5-fold, whereas superoxide dismutase-2 (SOD2) was upregulated 2.3-fold after 24 h of CM treatment . Upon H2O2 treatment, a significant reduction in the ROS level was observed not only in control cells with basal NKX3.1 expression but also in cells with ectopic NKX3.1 expression  levels  upon treatment with CM and H2O2, concurrent to the increase in SIRT1 expression/stability upon CM treatment, although the γ-H2AX(S139) levels were not increased upon treatment with H2O2. However, the antioxidant (LNAC) restored SIRT1 expression to its basal level in cells exposed to high concentrations of CM  level remained high upon CM treatment and was partially restored upon H2O2 treatment. CM treatment results in a significant amount of DNA damage, suggesting that the level of DNA damage is high in the inflammatory microenvironment but not in more highly oxidative conditions, in which the putative responsive pathways remain to be elucidated. On the other hand, because the relative SIRT1 mRNA level  level. Interestingly, we found that ATM(S1981) phosphorylation was not significantly increased upon CM treatment. LNAC only induced p-ATM(S1981) in control cells and not after CM exposure, suggesting that the reduced oxidative stress levels might trigger a cell cycle progression and DNA damage response, presumably via SIRT1-mediated p53 and NBS1 activations respectively. However, this hypothesis requires further investigation. We also examined whether apoptosis was induced in response to DNA damage, and found that the ATM-mediated response upon CM exposure did not generate caspase-3 cleavage in LNCaP cells, either with or without LNAC treatment  or normal media or H2O2  for a 2-week period. First, we analyzed the proteasomal degradation of NKX3.1, which correlated with exposure to increasing concentrations of CM , marginal upregulation of the antioxidant GPX2 (increase 1.3-fold) and downregulation of GPX3 (2.5-fold) after CM (100 pg/ml of TNFα) treatment . We also measured the intracellular ROS levels after the chronic treatments to gain insight into the changes in the oxidative conditions within the cells. Chronic CM exposure dose dependently correlated with an increase in the ROS level, but chronic H2O2 exposure maintained the ROS level close to the basal concentration  CM treatment. The proliferation rate was suppressed with either LNAC treatment (Figure in vitro, closely resembling animal studies [Overall, the inflammatory microenvironment is the major factor leading to sustained DNA damage in LNCaP cells, and the loss of NKX3.1 is an important contributing event. Therefore, the use of antioxidants might have a limited, but important ability to suppress the associated tumorigenic alterations, particularly in prostatic inflammation related cancer development. Upon chronic exposure of LNCaP cells to CM to mimic inflammation-like conditions, the loss of NKX3.1 leads to the deregulation of oxidative stress scavengers and contributes to increased DNA damage, eventually the prostate tumor progression Figure . The inf studies .Figure 6


The Fractionated Plasma Separation and Adsorption System Prometheus®  aims at being a supportive therapy as a bridge to transplantation or recovery in adults with liver failure. The system offers specific challenges when applied in children due to the large extracorporeal volume (700 to 750 ml). We therefore developed an adapted protocol for the application in children.3) were calculated from pretreatment and posttreatment serum concentrations. Primary and secondary patient outcome was evaluated.Priming of the blood circuit is performed using 2 l isotonic saline, whereas the plasma circuit, containing both adsorption devices, is filled with 2 U fresh frozen plasma or 400 ml stabilized solution of human plasma proteins. Next, for children with body weight (BW) <25 kg, a solution of 60 to 65% packed cells (PC) is infused in the inlet blood line at 40 ml/minute. The volume of PC needed is calculated based on the circuit priming volume and the maximum allowed extracorporeal blood volume of the child (= 8 ml/kg × BW). After the priming phase, blood and plasma flow are increased to at least 100 ml/ minute and 200 ml/minute, respectively, and dialysate flow is set at 300 ml/minute. Regional citrate anticoagulation is done with a calcium-free dialysate, while, eventually, heparin is added to the priming solution. Post treatment, the circuit volume is either not reinfused (BW <25 kg) or reinfused using isotonic saline (BW >25 kg), with a volume depending on the hydration status and the originally infused volume of PC. Reduction ratios  of urea, creatinine (Crea), bilirubin (bili), and ammonia  and 9 Fr Jugularis (n = 2)) were treated according to this protocol. In total, 19 sessions were executed using FX40 (n = 13), FX50 (n = 3), and FX60 (n = 3) dialysers during 6.5 ± 0.9 hours. Blood flow was 149 ± 45 ml/minute, albumin flow 226 ± 49 ml/minute, and ultrafiltration flow 432 ± 517 ml. RRs were 70 ± 15% (urea), 34 ± 14% (Crea), 44 ± 16% (bili), and 36 ± 10% (NH3). Primary survival rate was 100%. Four patients were transplanted (bridge to transplant) of which, however, one died within 30 days after discharge from the ICU. The fifth patient died due to primary disease 9 months after treatment, and the remaining three patients fully recovered (bridge to recovery).Eight children , 8.6 ± 5.9 years old (range 2 to 15.6 years), BW 32 ± 21 kg, GFR 71 ± 20 ml/minute/1.73 mIn conclusion, this adapted Prometheus® protocol is promising for the treatment of children with liver failure.


S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG) and catalytic triad (Ser88-Asp208-His235) of the esterase/lipase superfamily. The HydS14 sequence shows 46%–64% identity to 23 sequences from actinomycetes (23 α/β-hydrolases), has three conserved regions, and contains the novel motif (GY(F)SLG), which distinguishes it from other clusters in the α/β-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14) was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0–8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The Km and Vmax values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 μmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2–C6), displaying optimal activity with p-nitrophenyl-C4 (Kcat/Km = 11.74 mM−1·S−1). In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris.A thermostable esterase gene ( Following the lipolytic classification, this new esterase family was designated as family IX. Levisson et al. [Thermotoga maritima and classified it into family X. Lee et al. [et al. [et al. [Geobacilluskaustophilus HTA426, and Rao et al. [Thermoanaerobacter tengcongensis MB4, proposing the introduction of novel families  based on the sequences of EstA3 and other previously reported esterases. These proteins were designated as family XI (LipG), family XII (LipEH166), family XIII (Est30) and family XIV (EstA3).Lipolytic enzymes, including lipases, triacylglycerol hydrolases EC 3.1.1.3), esterases, and carboxylester hydrolases (EC 3.1.1.1) are among the most important groups of biocatalysts for biotechnological applications. Bacterial lipolytic enzymes can be classified into eight families based on conserved sequences, pentapeptide motifs (GXSXG) and biological properties [.1.3, estet al.  discovere et al.  screened [et al.  also fou [et al.  identifio et al.  reportedp-nitrophenol acyl esters. Esterases hydrolyze the ester bonds of water-soluble acyl esters and emulsified glycerolesters with short-chain acyl groups (≤C8), whereas lipases prefer water-insoluble acyl esters and emulsified substrates with long-chain acyl groups (≥C10) [Esterases and lipases are classified into two groups based on their substrate specificities toward groups ≤C, whereass ≥C10) . These e . These s (≥C10) ; the mots (≥C10) .Actinomadura sp. S14, from rubbish compost in Thailand and cloned a thermostable xylanase gene [Actinomadura to date, even though the phenotype of strain S14 on a tributylin agar plate suggested the presence of an esterase.Most lipolytic enzymes used in industry do not require cofactors are highly stable in organic solvents, and exhibit a wide substrate specificity and high stereoselectivity . HoweverActinomadura sp. S14, HydS14 showed 46%–64% identity and shares a pentapeptide motif with 23 sequences from actinomycetes, but HydS14 was cloned and expressed for the first time among them. The recombinant HydS14 was characterized as a new type of esterase in the lipase superfamily.This study focused on the cloning and characterization of a thermostable esterase from Actinomadura sp. strain S14 genomic library were screened for the formation of a clear zone on low-salt LB agar supplemented with 1% (w/v) tributylin, 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and 50 µg/mL kanamycin, as described in the S14) contained an insert of approximately 2.8 kb and the insert’s sequence was deposited into the DDBJ database under accession no. AB562501. The nucleotide sequence of the insert was analyzed using the NCBI sequence analysis tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), and it was determined that the pZErO-S14 insert includes an open reading frame (ORF) of 777 bp . This ORF encodes a polypeptide of 258 amino acids, with a predicted molecular mass of 26,604 Da. The ORF also possesses a GYSLG motif at positions 86–90, which is in agreement with the consensus sequence motif (GXSXG) of the lipase superfamily. HydS14 showed 46%–64% identity to α/β-hydrolases from a variety of actinomycetes, including genera Streptomyces, Catenulispora, Amycolatopsis, Saccharomonospora, Nocardiopsis, and Nocardia, (S. erythraea NRRL 2338 (YP_001103223). Interestingly, 3-oxoadipate enol-lactone hydrolases (EC 3.1.1.24), which are key enzymes in the β-ketoadipate pathway for the dissimilation of aromatic compounds, are included in this same group cluster [The esterase activities of approximately 15,000 transformants from an ocardia, . All of ocardia,  and sharocardia, . Region  cluster . α/β-Hyd cluster , includihydS14 gene that encodes a thermostable esterase from Actinomadura sp. S14, an organism from which we previously cloned a gene (xynS14) encoding a thermostable xylanase [E. coli was negligible. The effective expression of actinomycetes genes in Pichia has been previously reported [Pichia is dependent on the copy number of the gene in the genome (a higher copy number confers higher resistance to Zeocin), the transformants were screened on yeast extract peptone dextrose (YPD) plates containing 100, 1000, or 2000 µg/mL Zeocin. Twenty positive transformants, from the P. pastoris KM71H and X33 strains (which are resistant to 2000 µg/mL Zeocin) were selected and the levels of the expressed protein in the culture supernatants were compared. The esterase activity of the top transformant in strain X33 (218 ± 20 U/mL and 45 ± 1.5 U/mg) was higher than the top transformant in strain KM71H (128 ± 12 U/mL and 18 ± 0.8 U/mg) when grown at 30 °C for 144 h in 2 mL of BMMY medium supplemented with 2% methanol. Therefore, the transformant in strain X33 was used throughout this study. Using SDS-PAGE  to C16  in chain length as described in the pNP-butyrate (C4) (pNP-caproate (C6) to pNP-palmitate (C16). No activity was observed for substrates with a chain length of ≥C16. HydS14 exhibited a Km of 0.21 ± 0.02 mM and a Vmax of 37.07 ± 1.03 μmol/min/mg for pNP-butyrate. The Kcat/Km ratio was 11.74 ± 0.78 mM−1·S−1 and indicated that pNP-butyrate (C4) is the best substrate among the pNP-acyl ester examined  or a true lipase (no activity toward longer acyl esters).The substrate specificity of HydS14 was determined using ate (C4) , and theexamined . Pleiss v/v) to examine their effects, as described in the 2+, K+ and Ca2+ but was strongly inhibited by Fe2+ and Hg2+, as shown in p-chloromercuribenzoic acid. Phenylmethylsulfonyl fluoride (PMSF) strongly inhibited the enzyme’s activity, supporting the hypothesis that the serine residue in the conserved pentapeptide motif is the catalytic amino acid.HydS14 also hydrolyzed triacetin (C2) to tricaprylin (C8) and showed the highest activity with tributyrin (C4). These results indicate that HydS14 is an esterase and not a true lipase. Various detergents and solvents were incubated with HydS14 at final concentrations of 5% and 10%  and pZErO-2 (Invitrogen) were used as the host and vector, respectively, for the construction of a genomic library of strain S14. The pGem®-T Easy vector , E. coli TOP10 F’ cells (Invitrogen), and E. coli DH5α cells  were used for subcloning. Two expression systems were used for esterase gene cloning and expression: (1) pQE-80L  and E. coli Rosetta-gami B (DE3)  and (2) pPICZαA and P. pastoris strain X33 or strain KM71H using an EasySelect™ Pichia Expression Kit (Invitrogen). E. coli Rosetta-gami B (DE3) cells were grown in Luria-Bertani (LB) medium supplemented with 30 μg/mL chloramphenicol at 37 °C for 18–24 h. P. pastoris strain X33 or strain KM71H was grown on YPDS agar containing 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose, 1 M sorbitol, 100 μg/mL Zeocin and 2% (w/v) agar according to the provider’s manual. p-Nitrophenyl (pNP)-acyl derivatives and glycerol triacyl derivatives were purchased from Sigma-Aldrich . All other chemicals were of the highest grade available through commercial routes.et al. [Sau3AI. Digested DNA fragments of 2–10 kb were extracted using the Gel Extraction Wizard Kit (Qiagen), and these fragments were ligated into BamHI-digested pZErO-2 and transformed into E. coli TOP10 cells. The esterase activity of the bacterial transformants was confirmed on low-salt LB agar supplemented with 1% tributylin, 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), and 50 µg/mL kanamycin. The plates were incubated at 37 °C for 24–48 h, and positive transformants were detected by the formation of a clear zone. The positive colonies were selected, and their activities were reconfirmed using the same procedure. The plasmid harboring the esterase gene was extracted, and the size of the insert was confirmed. The nucleotide sequence of the insert was analyzed by primer walking, and the open reading frame (ORF) was analyzed using the NCBI sequence analysis tool (http://www.ncbi.nlmnih.gov/gorf.html). A homology search against the GenBank database was performed using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and multiple alignments were performed with the ClustalW program . The ORF of the esterase gene (hydS14) suggested that the mature esterase sequence is 777 bp. No signal peptide was predicted using the SignalP 4.1 Server . Phylogenetic trees of homologous hydrolase and esterase genes were constructed using the neighbor-joining algorithm in the MEGA 5.1 software with 1000 bootstrap sampling.The chromosomal DNA of strain S14 was extracted following the method of Kieser et al. , with a hydS14 fragment was amplified by PCR with KOD-Plus DNA Polymerase  in the presence of 5% (v/v) dimethyl sulfoxide (DMSO) directly from the genomic DNA of Actinomadura sp. S14. BamHI and PstI restriction sites were incorporated into the forward and the reverse primer sequences, respectively . The following PCR conditions were used: 1 cycle of 5 min at 98 °C, 35 cycles of 15 s at 98 °C, 1 min at 65 °C, and 1 min at 68 °C, and a final extension at 72 °C for 7 min. A poly A tail was added to the PCR products using 10X A-Attachment Mix (Toyobo). The PCR products were then purified and ligated into the pGem®-T Easy vector (pGem-hydS14). The hydS14 gene was subcloned into pQE-80L, which was then transformed into E. coli Rosetta-gami B (DE3) cells. The transformants were grown in LB supplemented with 50 μg/mL ampicillin at 37 °C for 2–3 h with shaking until the optical density at 600 nm reached 0.5–0.6. IPTG (100 μg/mL) was added to the culture, which was incubated for an additional 12–15 h. The cells were collected by centrifugation and disrupted by sonication. The cell debris was removed by centrifugation, and the resulting cell-free extract was subjected to SDS-PAGE to confirm the expression of HydS14, though no detectable band was observed. We then attempted to express the gene in P. pastoris. The hydS14 gene was amplified using the same procedures as described above except using the primers Hy-XhoF (5ʹ-CCGCTCGAGAAAAGAAGCAGGTTCGCGCGCACGGT-3ʹ) and Hy-XbaR (5ʹ-TGCTCTAGATGCGGCGGTCTCGTCGAGGAACGTTG-3ʹ); the XhoI and XbaI restriction sites are underlined. The PCR products were ligated into pPICZαA (pPIC-hydS14) and transformed into P.pastoris by electroporation using a Gene Pulser II  under the following conditions; 1.5 kV, 25 μF, 200 Ω and 4–6 ms. For efficient integration into the Pichia genome at the alcohol oxidase (AOX1) locus, pPIC-hydS14 was linearized with SacI prior to transformation. The digested pPIC-hydS14 was then purified and transformed into P. pastoris KM71H and X33 strains. Transformants from both hosts were grown on YPDS agar containing 100, 500, or 2000 mg/L Zeocin. By this scheme, the promoter for the AOX1 gene regulates the transcription of the hydS14 gene, and its expression is induced with methanol. The transformants were cultivated on BMGY medium (1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) yeast nitrogen base, 0.1 M potassium phosphate (pH 6.0), 0.4 μg/mL biotin, and 1% glycerol) at 30 °C for 48 h with shaking until the optical density at 600 nm reached 2–6. The yeast cells were aseptically harvested by centrifugation at 1200 rpm for 10 min, resuspended in 2 mL of BMMY medium (1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) yeast nitrogen base, 0.1 M potassium phosphate (pH 6.0), 0.4 μg/mL biotin, and 0.5% methanol) and incubated at 30 °C for 6 days with shaking. At 24 h intervals methanol was added to the culture to maintain a final concentration of 0.5%. The culture of each of transformant was centrifuged at 5000× g for 20 min to remove the cells. The supernatants were concentrated using a Vivaspin 20 concentrator  with a 10 kDa molecular weight cut-off membrane filter, and the resulting concentrate was absorbed onto a Ni-Sepharose 6 Fast Flow column . The enzyme was eluted with 50 mM Tris–HCl buffer (pH 8.0) containing 300 mM imidazole HCl. The enzyme was desalted using PD-10 columns  and subjected to SDS-PAGE to confirm the purity of the recombinant protein.The v/v) isopropanol at 4 °C for 1 h, and the gel was then washed twice with distilled water to remove the isopropanol [w/v) tributyrin agar gel containing 100 mM Tris–HCl buffer (pH 8.0) and kept at 60 °C for 3 h. Activity in a resolved band was observed as a clear zone against the white background of the gel but is shown as the reverse image in SDS-PAGE was performed using 12.5% polyacrylamide geld according to the method of Laemmli . The sampropanol . The wasp-nitrophenol liberated from the p-nitrophenyl-butyrate (C4) substrate at 410 nm. The enzyme reaction was performed at 50 °C for 5 min in 50 mM Tris–HCl buffer (pH 8.0) containing 0.4% Triton X-100, and 0.1% gum arabic. A final concentration of 0.2% SDS was added to terminate the reaction and put on ice. One unit of esterase activity was defined as the amount of enzyme that produced 1 μmol of p-nitrophenol per min at 50 °C. The extinction coefficients of p-nitrophenol were determined for every condition prior to each measurement. The background hydrolysis of the substrate was measured in a reference sample without the enzyme. The amount of protein was determined using the Bradford method [p-nitrophenyl-acyl esters with different acyl chains (p-nitrophenyl-acetate (C2) to p-nitrophenyl-caprylate (C8)). All the experiments were performed in triplicate. The kinetic parameters were calculated using Lineweaver-Burk plots.Esterase activity was determined by measuring the absorbance of d method . The subw/v) concentration) was preincubated at 60 °C for 15 min with magnetic stirring in a 50 mL flask with a stopper. After the addition of the purified enzyme, the total volume was adjusted to 30 mL, and 5 mL of each reaction mixture was withdrawn every 5 min. This aliquot was added to 25 mL titration cocktail flasks containing 10 mL of 95% (v/v) ethanol and 2–3 drops of 1% (w/v) thymolphthalein. Titration was performed with a burette titrator using 0.05 N NaOH until a light blue color appeared. The liberated fatty acids were calculated based on the amount of NaOH used to reach the titration end point using the following Equation:The activity of the esterase toward glycerol triacyl derivatives  was determined using a standard titration assay, with minor modifications . The subA reaction curve was created by plotting the quantity of liberated fatty acid over the time of the reaction, and the activity of the enzyme was determined from the slope of the linear portion. One unit was defined as the amount of enzyme that produced 1 μmol of fatty acid per min under the specified assay condition. The reaction was performed in duplicate for each substrate.v/v) of detergent or solvent. The residual activity was measured by comparing the enzyme activity in the presence of detergent or solvent to the enzyme activity in the absence of detergent or solvent. The effects of inhibitors and metal ions on esterase activity were measured by determining the residual activity after incubating the enzyme at 37 °C for 1 h with 1 mM of each inhibitor or metal ion. The enzymatic activity without any additive was defined as 100%. All the assays were performed in triplicate using p-NP-butyrate, as described.The optimum pH was measured using three different buffers at a final concentration of 100 mM with a pH range of 3.0–10.0  at 70 °C for 5 min. The optimum temperature was determined in 100 mM Tris–HCl buffer (pH 8.0) at different temperatures (30–90 °C). The pH stability was determined by measuring the remaining activity after incubating the enzyme at different pH values at 60 °C for 30 min. The purified enzyme was incubated at different temperatures from 50 to 80 °C for 150 min in 100 mM Tris–HCl buffer (pH 8.0), and the residual activity was measured. The effects of detergents and solvents on the esterase activity were determined by incubating the enzyme at 37 °C for 30 min in 100 mM Tris–HCl buffer (pH 8.0) containing 5% or 10% (hydS14) was cloned from an Actinomadura sp. S14 gene library. The encoded protein (consisting of 258 amino acids) contains a pentapeptide motif (GYSLG) and the catalytic triad (Ser88-Asp208-His235) of the esterase/lipase superfamily. HydS14 showed 46%–64% identity to 23 sequences from actinomycetes, including three conserved regions and a novel (GY(F)SLG) motif that distinguished the cluster from other clusters of the α/β-hydrolase structural superfamily. HydS14 with a 6X His tag (expressed in the plasmid pPICZαA-hydS14) was inducibly expressed with methanol in Pichia pastoris under the control of the AOXI promoter in a secreted protein form, consisting of a single protein that was not N-glycosylated. HydS14 demonstrated optimum activity at a temperature of approximately 70 °C and a pH of 8.0. The enzyme was stable at 50 and 60 °C for 120 min, with residual activity of more than 80% and over 90% and 50% activity in pH ranges of 6.0–8.0 and pH 9.0, respectively. HydS14 was tolerant to detergents and solvents, suggesting a potential for industrial applications.A novel esterase gene (


Many human diseases are correlated with the dysregulation of signal transduction processes. One of the most important protein interaction domains in the context of signal transduction is the Src homology 2 (SH2) domain that binds phosphotyrosine residues. Hence, appropriate methods for the investigation of SH2 proteins are indispensable in diagnostics and medicinal chemistry. Therefore, an affinity resin for the enrichment of all SH2 proteins in one experiment would be desirable. However, current methods are unable to address all SH2 proteins simultaneously with a single compound or a small array of compounds.In order to overcome these limitations for the investigation of this particular protein family in future experiments, a dipeptide-derived probe has been designed, synthesized and evaluated. This probe successfully enriched 22 SH2 proteins from mixed cell lysates which contained 50 SH2 proteins. Further characterization of the SH2 binding properties of the probe using depletion and competition experiments indicated its ability to enrich complexes consisting of SH2 domain bearing regulatory PI3K subunits and catalytic phosphoinositide 3-kinase (PI3K) subunits that have no SH2 domain.The results make this probe a promising starting point for the development of a mixed affinity resin with complete SH2 protein coverage. Moreover, the additional findings render it a valuable tool for the evaluation of PI3K complex interrupting inhibitors. Many human diseases are caused by imbalanced regulation of crucial cellular processes. Cell metabolism is regulated via a multitude of complex signal transduction cascades. Signals derived from extracellular stimuli are forwarded by phosphorylation events, which need to be strictly controlled. Dysregulation of these signaling processes can cause many human diseases such as cancer . Differe2 that was previously applied for a thermodynamic and structural study on SH2 domains [2 are available to determine potential linker modification sites. Another key feature of inhibitor 2 is its non-hydrolysable phosphotyrosine building block that was incorporated into the molecule without significant loss of biological activity. One of the inhibitor’s pentyl residues was chosen for linker modification, since it points outside the binding site. Molecular docking analysis was used to confirm similar target binding properties of probe 1 compared to inhibitor 2[1 (probe 1) is in accordance with the original inhibitor 2  the intensity ratio of SH2 proteins varies from 0.06% to 0.18%. This shows that the enrichment with probe 1 under optimized conditions with the mixed cell lysate increases the SH2 protein abundance by a factor of 43 to 87 . Since the utilized cell lines feature different protein profiles, a mix of these lysates offers a versatile pool of proteins for pull-down experiments. This cell lysate mix was used for protein kinase enrichment before and performed well, which led to the conclusion, that it might also contain a high variety of other signaling-related proteins such as SH2 proteins. Therefore, this mixed cell lysate was chosen for the first pull-down experiments  for the captured regulatory PI3K subunits. These values are comparable with the data published for several SH2 domain addressing inhibitors (3-50 μM) [1 via an SH2 domain. The regulatory PI3K subunits PI3KR1 and PI3KR2 contain a C-terminal and an N-terminal SH2 domain. The real IC50 values are likely smaller than the measured values for PI3KR1 and PI3KR2, since it is impossible to distinguish whether one of the two domains or both simultaneously interact with equal affinity with probe 1[1 a valuable tool for the investigation of PI3K subunit interactions and additionally underline the potency of probe 1 to bind SH2 domains.A competitive binding experiment was performed to validate the results obtained under pervanadate-depletion conditions  ,33,34. Ah probe 1. As expe1 selectively enriches SH2 proteins and allows for the identification of 22 of the 50 different SH2 proteins abundant in the chosen lysate with only one substance. The previously published IAP approach with the highest amount of captured SH2 proteins used 57 13-membered pY-peptides immobilized on magnetic beads and captured 45 SH2 proteins [1 captures two PI3 kinase complexes it can be applied for the investigation of the participating SH2 interactions and the evaluation of PI3K complex interrupting inhibitors. However, a more sufficient cell lysate for SH2 protein enrichment would be desirable.Probe proteins . TherefoA two-tailed student’s t-test assuming equal variance was performed with log transformed LFQ intensities to obtain p-values for each protein .The docking calculations were performed using AutoDock Vina and AutoDock Tools . All doc2 at 37°C using Roswell Park Memorial Institute 160 medium (RPMI1640) for the K562  and Colo205  cells, Dulbecco’s modified Eagle’s medium (DMEM) for OVCAR8  and SKNBE2  cells and Iscove’s modified Dulbecco’s medium (IMDM) for HeLa013 cells. All media were supplemented with 10% fetal bovine serum (FBS).Cells were cultivated in humidified air supplemented with 5% COFor pervanadate treatment, 100 mM sodium vanadate and 100 mM hydrogen peroxide solution were mixed in equal amounts resulting in a 50 mM pervanadate solution. The pervanadate solution was added to the cell culture medium to a final concentration of 100 μM and incubated for 25 min at 37°C.2, 150 mM NaCl, 0.8% NP-40, 1 mM dithiothreitol and 25 mM NaF with freshly added protease inhibitors and phosphatase inhibitors . Protein extracts were clarified by centrifugation for 20 min at 100,000 × g at 4°C and protein concentration was determined by the Bradford method.Cells were washed with cold phosphate buffered saline (PBS) and harvested by lysis using 50 mM Tris/HCl pH 7.5, 5% Glycerol, 1.5 mM MgCl1 ). For the dose response experiment, lysates were pre-incubated with the free compound for 30 min at 4°C. Lysates were incubated with beads for 30 min at 4°C. Subsequently, the beads were washed with 1× CP buffers containing 0.4% and 0.2% NP40 and 10% glycerol. Bound proteins were eluted with 2× NuPAGE LDS Sample Buffer  and eluates were reduced and alkylated by 50 mM dithiothreitol and 55 mM chloroacetamide. Samples were then run into a 4–12% NuPAGE gel  for about 0.5 cm to concentrate the sample prior to in-gel tryptic digestion. In-gel trypsin digestion was performed according to standard procedures.SH2 probe pulldowns were performed as described previously . Briefly18-AQ 5 μm resin, Dr. Maisch, Ammerbuch, Germany) at a flow rate of 5 μL/min in 100% solvent A (0.1% formic acid in HPLC grade water). After 10 min of loading and washing, peptides were transferred to an analytical column  and separated using a 210 min gradient from 4% to 32% of solvent B  at 300 nL/minute flow rate. The LTQ Orbitrap XL was operated in data dependent mode, automatically switching between MS and MS [Nanoflow LC-MS/MS was performed by coupling an Eksigent nanoLC-Ultra 1D +  to a LTQ-Orbitrap XL ETD . Peptides were delivered to a trap column  for peak detection and quantification  MS/MS spLFQ intensities of 0 (in control replicates) were replaced with the 1% percentile of the LFQ intensities of this respective measurement for data visualization.50: Half maximal inhibitory concentration; LC-MS/MS: Liquid chromatography tandem mass spectrometry.ATP: Adenosine triphosphate; SRC: Proto-oncogene tyrosine-protein kinase SRC; SH2: SRC homology 2; AKT: Serine/threonine-protein kinase AKT; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; JNK: c-Jun N-terminal kinase; PI3K: Phosphoinositide 3-kinase; GST: Glutathione-S-transferase; IAP: Inhibitor affinity purification; EGFR: Epidermal growth factor receptor; PI3KCA/B: Phosphoinositide 3-kinase catalytic subunit A/B; PI3KR1/2: Phosphoinositide 3-kinase regulatory subunit 1/2; ABL1: Abelson murine leukemia viral oncogene homolog 1; LFQ: Label free quantification; ICThe authors declare that they have no competing interests.The manuscript was written through contributions of all authors. All authors read and approved the final manuscript.Proteomic data. List of captured SH2 proteins.Click here for fileProteomic data. Pull-down optimization.Click here for fileDetailed experimental procedures. Experimental procedures and analytical data of the compound synthesis and supplementary figures and tables.Click here for fileProteomic data. Data set of the pervanadate depletion experiments.Click here for fileProteomic data. Data set of the dose response experiments.Click here for file


C) comprises a natively unstructured N-terminal domain, including a metal-binding octarepeat region (OR) and a linker, followed by a C-terminal domain that misfolds to form PrPSc in Creutzfeldt-Jakob disease. PrPC β-endoproteolysis to the C2 fragment allows PrPSc formation, while α-endoproteolysis blocks production. To examine the OR, we used structure-directed design to make novel alleles, ‘S1’ and ‘S3’, locking this region in extended or compact conformations, respectively. S1 and S3 PrP resembled WT PrP in supporting peripheral nerve myelination. Prion-infected S1 and S3 transgenic mice both accumulated similar low levels of PrPSc and infectious prion particles, but differed in their clinical presentation. Unexpectedly, S3 PrP overproduced C2 fragment in the brain by a mechanism distinct from metal-catalysed hydrolysis reported previously. OR flexibility is concluded to impact diverse biological endpoints; it is a salient variable in infectious disease paradigms and modulates how the levels of PrPSc and infectivity can either uncouple or engage to drive the onset of clinical disease.The cellular prion protein (PrP C), encoded by the Prnp gene, is displayed on the cell surface by a glycophosphatidylinositol (GPI) anchor and serves a precursor role, undergoing a change from a mainly alpha-helical structure to the beta-rich conformation of PrPSc during disease. Its function is debated such that it could be involved in neuroprotection , two hexarepeats, five tandem repeats of eight amino acids forming an octarepeat region (OR), a hydrophobic linker region sometimes referred to as the ‘HD’ (hydrophobic domain) and a C-terminal globular domain  and bovine spongiform encephalopathy (BSE) are transmissible neurodegenerative disorders. The cellular form of the prion protein , two having particular relevance here. The first concerns the crucial role of endoproteolytic events. One important misfolded protein species in AD is the amyloid beta peptide, Aβ, this being liberated by the concerted actions of the endoproteases called β- and γ-secretase on the beta amyloid precursor protein, APP. Conversely, α-secretase processing of APP by a disintegrin and metalloproteases (ADAM) generates a benign peptide called P3 and prevents amyloid formation. Notably, PrPvents Fig; α-cleavL—have proven difficult to study  is natively unstructured but that different stoichiometries of metal binding can impart different structures upon the OR  that could encode the conformationally constrained proteins  analysis of metal protein complexes, we identified residue substitutions in ORs 2–5 that constrained PrP to favour component 1 or 3 binding geometry  , S1 PrP (S1–29), S3 PrP (S3–27) and S3.F88W PrP (F88W-5). Cell surface proteins were biotin-labelled using a membrane-impermeable reagent and trypsin-digested following specific chase times . The mutn = 5), but a series of experiments modulating these baseline values by adding chelators or metals or by altering cell plating density failed to alter C2 formation by the S3 or WT PrP alleles  generated by the binding of copper to the PrP OR , there was no effect on the cleavage of S3 PrP. However, if compounded into triple or quadruple mutations (F2F3F4 and F1F2F3F4), C2 production was attenuated. There was no visible influence of the phenylalanine residue substitutions on PrP with WT or S1 versions of the OR, and cleavage to the C1 fragment was maintained in cells expressing all mutation variants -35 mouse were analysed, the ratios of C2 fragment to C1 or full-length PrPfered Fig. In the fered Fig. Additioet al, et al, et al, et al, Although increased levels of C2 fragments occur in prion disease states  mice exhibit a low level of expression .Outside of the CNS, independent lines of PrP knockout mice exhibit a demyelinating polyneuropathy (DMP) , with increased expression level being a parameter that normally decreases incubation times  mice .We next assessed the responses of TgPrPS1)-17 and TgPrP(S1)-19 mice upon challenge with infectious prions using the RML isolate of mouse-adapted scrapie. As presented in Table-17 and Tet al, et al, C substrate and can result in altered selection pressures that will facilitate the emergence of new strains -17 and TgPrP(S3.F88W)-35 animals were also challenged with prions from cases of natural scrapie and classical BSE. Animals were held for over 400 days without signs of clinical disease  mice, indicating that expression of the S3.F88W PrP allele did not favour the appearance of a new prion strain with different pathological properties. Incubation times were longer than for direct passage into WT mice (164.3 ± 3.5 (SEM) versus 155.9 ± 2.0 days ) and were consistent with a drop in titre measured by other techniques, as presented below.In terms of neuropathology, RML prion-challenged TgPrP(S1) and TgPrP(S3.F88W) exhibited reduced levels of deposition of protease-resistant PrPiosis Fig. They dirions Fig. InteresSc species: this inventory included net levels of protease-sensitive and protease-resistant (r) forms of PrPSc (rPrPSc), as well as PrPC levels in fractions at the top of the gradients  and TgPrP(S3.F88W) animals , revealed subtle distinctions in the PrPSc populations in the brains of RML-infected animals after PK treatment (Sc derived from RML-infected WT (Prnpa), TgPrP(S1)-17 and TgPrP(S1)-19 mice behaved in a similar manner with an increased D/N value after PK treatment, whereas PrPSc from infected TgPrP(S3.F88W)-14 and TgPrP(S3.F88W)-35 mice exhibited a drop in ratio. The assay used to obtain these data uses the 8H4 antibody for PrP capture and a Europium-labelled version of the 12B2 antibody for detection . As alterations were not detected in the C-terminal protease-resistant core of PrPSc  mice.We carried out further analytical chemical procedures to increase our understanding of the pathogenic process in TgPrPS1) and TgPrPS3.F88W) mice. We separated protein species on the basis of size by velocity gradient fractionation of infected brain samples performed in the presence of the detergent sarkosyl. Next, the conformation-dependent immunoassay (CDI), which provides a signature of global protein folding via the accessibility of antibody epitopes, was used to assess the gradient fractions. Using these two technologies, and by performing analyses of gradient fractions in the presence or absence of proteinase K, we compiled an inventory of oligomeric PrP and TgPr.F88W micI assays . The sed animals . A differeatment . PrPSc dSc, with infected TgPrP(S1) and TgPrP(S3.F88W) mice at disease endpoint both having lower levels than those of WT mice  and TgPrP(S3.F88W) mice, respectively. Figures for the analogous reductions in protease-resistant PrPSc averaged across Tg lines of the same allelic type were 4.0× and 4.6×, respectively. These lower values for PK-sensitive PrPSc (and rPrPSc) were also paralleled by a drop in infectious titre as established by a homologous serial passage, which revealed a prolongation of incubation times . Secondly, brain homogenates from primary passages into TgPrP(S1) and TgPrP(S3.F88W) mice generated significantly lower spot counts by the standard scrapie cell assay -17, TgPrP(S1)-19 and TgPrP(S1)-39 lines plotted to a different regression from mice expressing different levels of the WT PrPC allele (Prnpa), while the TgPrP(S3.F88W)-14 and TgPrP(S3.F88W)-35 lines behaved in yet a third distinctive manner  and TgPrP(S3.F88W) mice; instead, repeated measures revealed drops in the quantities of protease-sensitive PrPSc, rPrPSc, infectious titre and associated neuropathology. Lastly, lower prion titre by SSCA  yet shorter incubation times (TableP = 2.44E-19 and 1.75E-18 in TgPrP(S3.F88W)-14 and TgPrP(S3.F88W)-35 mice, respectively) versus WT mice indicates an uncoupling effect such that these Tg animals respond to pathologic forms of PrP differently from their WT counterparts  mice stand in apparent contrast to this effect. To explore this effect and to accommodate the post-translational reduction in PrPanner Fig, indicatparts Fig, as discet al, et al, et al, in vivo. With recombinant PrP  in RK13 cells or Tg mice resulted in a ∽3.5-fold elevated level of a ∽20–21 kDa C2 fragment. A reciprocal trend for decreased C2 production by S1 alleles  mice and Tga20 mice expressing WT PrPC . Thus, S3 PrP is likely processed by the same protease that processes WT PrPC.Although prolines play a key role cuing the S3 PrP allele for C2 proteolysis Figs and 2C, et al, et al, Enhanced C2 generation by the S3 allele appears to reflect a facet of N-terminal domain function that is not related to the nature of amino acid residue side chains, but that is impacted by the flexibility of the polypeptide backbone. Proline is a unique component of polypeptides with partial double-bond character in the carbon–nitrogen backbone (between the nitrogen of the ring structure and proceeding carbon atom with a carbonyl group) that restricts free rotation. While two GQ > PP substitutions in the S1 PrP allele had no effect upon C2 production Figs and distet al, et al, C remains PK sensitive -17 and TgPrP(S1)-19 lines, PrPint Table and spotint Table were depC in uninfected TgPrP(S3.F88W)-14 and TgPrP(S3.F88W)-35 mice are increased 0.7× over TgPrP(S1)-17 and TgPrP(S1)-19 mice, while the ratio of C2 production changes by greater than three-fold -17 and TgPrP(S1)-19 mice. Two variables are relevant in drawing these comparisons: the levels of full-length PrP-17 and Tint Table. Our stuint Table). To resint Table. Future Sc has been seen previously, and at one stage, it was used to argue against the validity of the prion hypothesis that the infectious agent is composed of misfolded PrP , annealed into the full-length PrP sequences in an existing pCDNA3 plasmid using T4 DNA ligase (New England Biolabs), and correct full-length PrP sequences were then moved into another vector pBudgfp by digestion with XbaI and HindIII (New England Biolabs), followed by gel extraction and ligation. For phenylalanine substitutions into the hydrophobic domain, proline substitutions into the octarepeats  and H95A mutations, primers were designed as per the GeneTailor Site-Directed Mutagenesis kit (Invitrogen) and the mutagenesis reaction was carried out as per the manufacturer's protocol. One microlitre of each reaction was transformed into competent DH5α cells (Invitrogen), and plasmids were isolated using a miniprep kit (Qiagen). Correct plasmids were confirmed by sequencing.To create novel alleles in the PrP OR equivalent to the synthetic peptides  examined by electron paramagnetic resonance (EPR) spectroscopy , the sequences encoding the N-terminal portion (with the mutations included) of PrP and part of the 5' UTR were synthesized by GenScript Inc. The cassettes encoding the OR-encoding sequences of interest were excised from the plasmid provided by GenScript using 2 levels. Cells of approximately 90–100% confluency were transfected using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. RK13 cell lines stably expressing wild-type (WT) PrP (WT-10) and mutant S1 (S1–29), S3 (S3–27) and S3.F88W (F88W-5) PrP were created by transfection with plasmids encoding the proteins, and clones were selected using zeocin (Invitrogen). Clones isolated with cloning rings (Scienceware) were amplified, lysed with RIPA buffer  and analysed by Western blot for PrP expression.Rabbit kidney epithelial (RK13), neuroblastoma (N2a), cured SMB (SMB-PS), human kidney epithelial (HEK) and human neuroblastoma (SH-SY5Y) cells were maintained in Dulbecco's modified Eagle's medium  supplemented with 10% foetal bovine serum  and penicillin/streptomycin (Invitrogen) at 37°C and 5% COXho ‘half-genomic’ vector  fragments were cloned into pCR2.1.TOPO (Invitrogen), and sequences were verified. DNA from TOPO clones was digested using XhoI (New England Biolabs), isolated by gel electrophoresis and purified with a gel extraction kit (Qiagen). The MoPrP.Xho vector was digested with XhoI, treated with calf intestinal alkaline phosphatase (New England Biolabs), phenol:chloroform-extracted and ethanol-precipitated or purified with the Ultraclean Gel Spin DNA Purification Kit (MoBio Laboratories). After ligation of digested fragments, the sequenced DNA was amplified and purified using the Endo-Free Maxiprep Kit (Qiagen). To prepare for injection, the DNA was digested with NotI and purified using the gel extraction kit (Qiagen) or Ultraclean Gel Spin DNA Purification Kit (MoBio Laboratories). The DNA was then injected into the pronuclei of mouse FVB embryos by the University of Calgary Transgenic Services.For the generation of transgenic mice, sequences encoding WT PrP and mutant S1 and S3.F88W PrP were inserted into the MoPrP.et al, g for 5 min and carefully layered onto the top of the 10% to 45% sucrose gradient prepared in PBS, pH 7.4, containing 1% sarkosyl. Ultracentrifugation was performed at 237,000 g for 73 min at 5°C in an Optima TL ultracentrifuge (Beckman) equipped with a Beckman SW 55 Ti rotor. After the centrifugation, fractions were collected from the bottom of the tube. The CDI assay was similar to previous descriptions  of brain, spleen, heart, kidney, liver and lung were prepared in PBS complemented with Complete Mini Protease Inhibitor (Roche) using a homogenizer  at 4°C. Sucrose gradient and the conformation-dependent immunoassay (CDI) were performed as previously described  membranes. Blots were immediately incubated with PrP antibody Sha31  or were blocked with 5% skim milk in 0.1% TBST for 1 h at room temperature and incubated with primary antibody at 4°C overnight , VRQ61 1/5,000, and 2D6 1/10,000, 1A6 1/5,000). 9A2 and 12B2 were gifts from Dr. Jan Langeveld  for 1 h at 37°C. The reaction was stopped with 2 mM phenylmethanesulfonyl fluoride (PMSF) and centrifuged at 13,000 rpm for 1 h at 4°C. The pellets were resuspended in 15 μl PBS and 3 μl of 6× SDS loading buffer was added before boiling. If samples were to undergo further PNGaseF digestion, the pellets were resuspended in 15 μl of 2% SDS and heated at 100°C for 10 min. Two microlitres of 10% NP-40, 2 μl of 10× G7 Reaction Buffer and 1 μl of PNGaseF were added, and the reaction was incubated at 37°C for 2 h. The reaction was stopped by adding 6× SDS loading dye and boiling. Samples not PK digested were digested by PNGaseF according to the manufacturer's protocol, overnight at 37°C with 0.2 μl of PNGaseF. g for 5 min, and the supernatant was collected. Equal amounts of brain homogenate were added to increasing concentrations of guanidine hydrochloride (GdnHCl) (Sigma), and the samples were shaken at 800 rpm for 2 h at room temperature. The concentration of GdnHCl was diluted to 0.4 M using RIPA buffer, and the total volume of each sample was made equal by adding 0.4 M GdnHCl. Samples were treated with 50 μg/ml PK for 1 h at 37°C, and the reaction was stopped using PMSF . The samples were centrifuged at 100,000 g at 4°C, the pellet was resuspended in PBS, and 6× SDS loading buffer was added.The 10% brain homogenate was centrifuged at 700ad libitum access to food and water. Male and female FVB mice 4–7 weeks of age were randomly assigned for inoculation with RML (source S. Prusiner), natural scrapie from a Canadian ARQ/ARQ sheep , classical BSE from a Canadian animal , or uninfected WT mouse brain homogenate (control). Approximate sample sizes for each group were determined by previous inoculation experiments in the literature. Thirty microlitres of 1% brain homogenate in PBS was injected using a 25-gauge needle into the parietal lobe of each mouse. The inoculum was heated for 20 min at 80°C before inoculations were performed. After inoculations, mice were checked for symptoms of prion disease, including head tilt, ataxia, extension, clasp, weight loss and stupor; initial diagnosis of clinical disease onset was performed by animal staff performing health monitoring. Animals euthanized due to non-prion disease-related illness or injury were excluded from analyses. Animal experiments were performed in accordance with the Animal Care Committee of the University of Alberta, protocols AUP00000356 and AUP00000357.Animals were housed in ventilated cages with a 12-h light/dark cycle and were given Sc using SAF83 antibody. Glutaraldehyde-fixed samples of sciatic nerve were processed as described previously  were used for toluidine blue staining, and ultrathin sections were used for electron microscopy. The ultrathin sections were mounted on formvar-coated copper grids and contrasted with uranyl acetate and lead citrate. The samples were viewed in a Jeol JEM-2100 electron microscope at an acceleration voltage of 120 kV and a magnification range of 1,500× to 4,000×. Electron micrographs were recorded on a Gatan Erlangshen ES500W CCD camera.t-tests were used for comparisons of these sample groups with no a priori predictions for the performance of the PrP alleles. For incubation time data with n < 6 where normal distribution could not be confirmed, we used a Mann–Whitney test. Statistical tests were performed using InStat and GraphPad Prism software with P-values < 0.01; < 0.001 and < 0.0001 represented by 2, 3 or 4 asterisks, respectively. Exact P-values are reported in the main text or the figure legends and were calculated using GraphPad Prism and Microsoft Excel.Normal distribution of data was confirmed by a Kolmogorov–Smirnov test, and unpaired, two-tailed Student's


N-ethylmaleimide-sensitive factor-attachment protein receptors (ER–Golgi SNAREs) syntaxin 5 (Syx5) in neuronal cells. Quantitative as well as qualitative data are presented here to verify the upregulation of Syntaxin 5 (Syx5) under ER and Golgi stresses in hippocampal neurons. Changes in the processing of β-amyloid precursor protein (βAPP) under ER stress were analyzed by immunological assays. In addition, our data shows the specific increase of Syx5 expression under ER and Golgi stresses. Interpretation of our data and further extensive insights into the role of Syx5 in βAPP processing under organelle stress can be found in "ER and Golgi stresses increase ER-Golgi SNARE Syntaxin5: Implications for organelle stress and βAPP processing" This paper reports the data for the effects of organelle stresses on the ER–Golgi-soluble  Specifications tableValue of the data•Data show the effects various stress inducers on the expression and intracellular localization of ER–Golgi SNARE Syx5 in hippocampal neurons.•Data obtained by immunological assays show the effects of ER stress inducers on the βAPP processing in primary culture neurons.•The data provided here may inform many researchers that are investigating neurodegenerative diseases of the effect of organelle stress in mammalian cells.1Here we showed quantitative as well as qualitative data concerning the effects of various ER and Golgi stress inducers on the expression and the localization of ER–Golgi SNARE proteins in hippocampal neurons. The data presented here provides important information on the use of various toxins that induce organelle stress in primary culture neurons. Syx5 is a member of ER–Golgi SNAREs and is a major player in the transport processes in the early secretory compartment 1.1Tunicamycin (Tm), Hoechst 33342, and brefeldin A (BFA) were purchased from Sigma-Aldrich Chemical Co. ; monensin and nigericin were obtained from Merck . Monensin, nigericin, and BFA were dissolved in methanol. Staurosporine (STS) and thapsigargin (Tg) were also purchased from Merck and dissolved in dimethyl sulfoxide. A protease inhibitor cocktail was purchased from Wako Chemicals . All other reagents were of the highest grade available, unless otherwise noted.1.2Mouse anti-Syx5 monoclonal antibody (clone 1C5) was prepared as described previously 1.34/cm2 in either a glass-bottomed 35-mm dish, in polyethyleneimine-coated 96-well plates , or in 12-well plates  and cultured in DMEM (Sigma-Aldrich) containing 10% fetal bovine serum at 37 °C with 95% air, 5% CO2 in a humidified incubator. After 24 h of in vitro culture, the medium was replaced with serum-free, DMEM-containing GlutaMax and 2% B-27 Supplement  either with or without 2 µM Ara-C (Sigma-Aldrich) and used after 13–16 days of in vitro culture.Pregnant Wistar Kyoto rats were obtained from Japan SLC, Inc. . All of the experimental procedures using animals were approved by the Experimental Animal Ethics Committee of the Kyorin University School of Medicine, and performed in accordance with the guidelines for handling laboratory animals. Primary rat hippocampal cultures were prepared as described previously 1.4The sequences and preparation of Syx5 siRNA#1 (R424442) and control siRNA were described previously 1.5Biochemical analyses were carried out as described previously 1.6Immunocytochemical analyses were carried out as described previously 1.72+ concentration ([Ca2+]i) was measured. Fura2 ratio imaging analysis was performed using an ARGUS50 Ca2+ imaging system  and an inverted microscope  equipped with a CCD camera . Fluorescent images were taken at excitation wavelengths of 340 and 380 nm and were acquired every 10 s. Cells were treated with 1 μM Tg at the time shown by arrows. The ratio of Fura2 fluorescence (F340 nm/F380 nm) was calculated and plotted against time, and the values from single cells were averaged at each time point and expressed as the mean±SD (n=10). SERCA inhibitor, Tg induced a transient increase in [Ca2+]i.Hippocampal cells were treated with Fura2-AM  for 25 min, washed, and allowed to stand for 20 min in normal medium. After changing the medium to HEPES containing Hank’s balanced salts solution, the intracellular Ca1.8Cell viability and cytotoxicity of cells were measured with a CellTiter-Glo Luminescent Cell Viability Assay and CytoTox-Glo Cytotoxicity Assay systems (Promega) according to the manufacturer’s instructions. Luminescence was measured using a luminometer .1.9g for 10 min; the resultant supernatant fraction was used as the enzyme-linked immunosorbent assay (ELISA) sample. Aβ peptides secreted into the culture medium (100 μL) were quantified using a sandwich ELISA specific for either rat Aβ1-40 or Aβ1-42 according to the manufacturer’s instructions (Takara Bio).Hippocampal neurons were cultured in 12-well plates (in 2 mL of medium) for 13 days before treatment. After the medium was replaced with fresh medium, the cells were treated with monensin or nigericin for 16 h. The culture medium was collected, and cell debris was removed by centrifugation at 15,0001.10t-test or ANOVA was used to determine the statistical significance of differences between values.The enhanced chemiluminescence signals from western blot analyses and the amount of each PCR product were analyzed using MultiGauge software . Statistical analyses were performed using GraphPad Prism software . Data are presented as mean±SEM unless noted. The number of samples examined is indicated in an inset within each figure. A n=11-12). Data sets were subjected to one-way ANOVA and with Dunnet’s post-hoc tests, ***P<0.0001.Data for 2+ imaging as described in Materials and Methods. Cells were treated with 1 μM Tg at the time shown by an arrow. Ratio of Fura2 fluorescence (F340 nm/F380 nm) were calculated and were plotted against time and the values from single cell were averaged at each time point and expressed as mean±SEM (n=10). SERCA inhibitor Tg induced transient increase in [Ca2+]i but returned to the basal level of [Ca2+]i. (B) Hippocampal neurons were treated with siRNA for Syx5 (R424442) and cell extracts were subjected to SDS-PAGE and western blotting with anti-Syx5 (1C5). Representative images are shown. (C) Densitometric analysis of western blotting in (B). The amount of each Syx5 isoforms was quantified, and the values are expressed as the mean percentage of the control siRNA-treated cells (n=28). * P<0.05 and *** P<0.0001 vs. control, as determined by a t-test.Data for P<0.05 and ** P<0.001 vs. vehicle control, as determined by a t-test. BFA treatment also induced de novo syntheses of ER–Golgi SNARE Syx5. (C) Effect of ER stress inducers on the intracellular localization of Syx5 isoforms and on the morphology of the Golgi apparatus. 16DIV hippocampal neurons were fixed and stained for Syx5 or GM130 after exposure to 1 µM Tg or 2 µg/mL BFA for 16 h. Representative images are shown. Tg and BFA treatments induced fragmentation of the Golgi as assessed by the localization of Golgi matrix protein GM130 (green). Dispersion of the Golgi apparatus observed in BFA-treated cells shows that BFA had a stronger effect than Tg on Golgi structure. Blue, nuclei stained with Hoechst 33342; red, Syx5 stained with monoclonal antibody 1C5 with a Cy3-conjugated secondary antibody; green, Golgi apparatus stained with monoclonal antibody against Golgi marker GM130 with an Alexa Fluor 488-conjugated secondary antibody.Data for n=9). Significant accumulation of endogenous βAPP holoproteins was observed in BFA-treated, but not in Tg-treated cells as in NG108-15 cells n=12). ***P<0.001 vs. vehicle, as determined by a t-test. Secretion of both Aβ peptides was markedly suppressed by induction of ER stress.Data for Data for n=26) or 2 µg/mL BFA (n=9) for 16 h, and the cell extracts were subjected to SDS-PAGE and western blot analysis. Western blots showing amounts of indicated Syxs were analyzed densitometrically (B). (C) Culture neurons were treated with 0.1 µM nigericin or 1 µM monensin for 16 h, and the cell extracts were subjected to SDS-PAGE and western blot analysis. Expressed amounts of Syx6 and Syx1A were analyzed densitometrically (D). *P<0.05 vs. vehicle control, as determined by a t-test (n=10–14). Among Syxs examined, both ER and Golgi stressors did not induce significant increase in the expression of Syx6 nor Syx1A.Data for