News Article | September 28, 2016
If not stated otherwise, all chemicals and reagents were obtained from Sigma Aldrich. Restriction enzymes were obtained from New England Biolabs. The AquaMet catalyst was purchased from Apeiron Synthesis S.A. All plasmids used in this study are collated in Supplementary Table 1. To construct the periplasmic expression vector for SAV, the gene for T7-tagged SAV was amplified by polymerase chain reaction (PCR) from pET-11b-SAV31 using primers 1 and 2 (Supplementary Table 2) to add the 21 amino acid OmpA signal peptide (MKKTAIAIAVALAGFATVAQA) to the N terminus of SAV. The PCR product was digested with restriction enzymes NdeI and BamHI, gel purified and ligated into the target vector pET-30b(+) (Merck Millipore) pre-treated with the same enzymes. The resulting expression vector, designated pET-30b-SAVperi, carries the gene for the OmpA::SAV fusion protein under the control of a P promoter. To construct a comparable cytoplasmic expression construct, the gene for T7-tagged SAV from pET11b-SAV was PCR-amplified without adding additional amino acids using primers 3 and 2 (Supplementary Table 2) and subsequently cloned into pET-30b(+) by restriction digest (NdeI and BamHI) and ligation, resulting in plasmid pET-30b-SAVcyto. All strains used in this study are summarized in Supplementary Table 3. A strain for periplasmic expression was constructed that combines the ease of library generation with the compatibility with the T7-expression system. Therefore, the gene of the T7 RNA polymerase was integrated into the chromosome of E. coli TOP10 (F− mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ−, Thermo Fisher Scientific) using the λDE3 Lysogenization Kit (Merck Millipore). The resulting lysogen was designated E. coli TOP10(DE3). E. coli TOP10(DE3) containing the respective expression plasmid for SAV (pET-30b-SAVperi or pET-30b-SAVcyto) was cultivated in a Luria–Bertani (LB) medium (50 ml)32 supplemented with kanamycin (50 mg l−1) in shake flasks (500 ml, 37 °C, 200 r.p.m.). An LB pre-culture was diluted 1:100 in fresh medium and incubation was performed until an optical density at 600 nm (OD ) of about 0.5 was reached. Subsequently, the cultivation temperature was lowered to 20 °C, and the expression of SAV was induced by addition of isopropyl-β-d-thiogalactopyranoside (IPTG, 50 μM) and cells were harvested by centrifugation (6,000 r.c.f., 2 min) after four hours of induction. The cell pellet was further processed either for fractionation to analyse the cellular protein content or for flow cytometry. To separate the periplasmic and the cytoplasmic fraction of cellular proteins, the PeriPreps Periplasting Kit (Epicentre Technologies) was used. For in-gel staining of SAV, biotin-4-fluorescein (10 μM) was added to the respective fractions before sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) analysis. Owing to the binding to the biotinylated fluorescent dye, SAV can be visualized under ultraviolet light in the gel (before staining with Coomassie blue). Owing to its high stability, SAV remains tetrameric and fully functional even under the otherwise denaturing conditions of SDS–PAGE. To stain SAV in whole cells of E. coli, the pellet of a shake-flask expression culture was resuspended in phosphate buffer saline (PBS32) to a final OD = 1 before the addition of Atto-565-biotin (2 μM, Atto-Tec GmbH). Cells were incubated on ice (30 min) and subsequently subjected to three wash cycles by centrifugation (6,000 r.c.f., 2 min) and resuspension of the pellet in PBS. The resulting cell suspensions were analysed on a BD LSR Fortessa SORP (BD Biosciences) using a 561-nm laser for the excitation of Atto-565-biotin and a 610/20BP-600LP filter combination for analysis of the emitted fluorescence signal (peak area). The histograms displayed comprise data of 100,000 ungated events for each sample. For expression of SAV (and mutants thereof) in the 96-deepwell format, LB medium (500 μl) supplemented with kanamycin (50 mg l−1) was inoculated from a single colony and grown until stationary phase (37 °C, 300 r.p.m., 50-mm shaking amplitude). This pre-culture (aliquot of 30 μl) was used to inoculate a main culture (1 ml) in a modified ZYM-5052 medium33 that lacked lactose as auto-induction agent, but contained kanamycin (50 mg l−1). Induction was performed by the addition of IPTG ([IPTG] = 50 μM) and continued for four hours before harvesting. Subsequently, a fraction of the cultures (100 μl) was set aside for OD determination and the remaining suspension was subjected either to ICP-OES quantification or to metathesis with whole cells. The activity of the artificial metalloenzyme biot-Ru–SAV was quantified in the reaction buffer (100 mM sodium acetate, pH 4.0, 0.5 M MgCl , 2.5% DMSO) both in the presence and absence of 10 equiv. of glutathione (GSH or GSSG; 500 μM) relative to the amount of the cofactor biot-Ru (50 μM). The reaction mixture (total volume of 200 μl) was incubated shaking (16 h, 37 °C) and, after the reaction, 800 μl of methanol containing 2-phenylethanol (final concentration of 100 μM) as an internal standard was added. After centrifugation (5 min, 21,000 r.c.f., 4 °C), 500 μl of the supernatant were diluted with 500 μl of de-ionized water and the samples were subjected to UPLC analysis. UPLC analysis was performed using a Waters H-Class Bio using a BEH C18 1.7 μM column and a flow rate of 0.6 ml min−1 (eluent A, 0.1% formic acid in water; eluent B, 0.1% formic acid in acetonitrile; gradient at 0 min: 90% A, 10% B; at 0.5 min: 90% A, 10% B; at 2.5 min: 10% A, 90% B; at 3.5 min: 90% A, 10% B; at 4.5 min: 90% A, 10% B). The ultraviolet signal at 210 nm was used for quantification, and concentrations of the metathesis product umbelliferone 2 (retention time of 1.38 min) were determined on the basis of a standard curve with commercially available umbelliferone (Sigma Aldrich). To quantify the ruthenium content of cells, cultures from 96-deepwell plates were pelleted by centrifugation (3,220 r.c.f., 12 min, 4 °C) and resuspended in ice-cold Tris/HCl-buffer (1 ml, 50 mM, pH 7.4, 0.9% (w/v) NaCl) containing biot-Ru (10 μM). After incubation on ice (30 min), the cells were spun down (1,260 r.c.f., 8 min, 4 °C), the supernatant-containing excess cofactor was discarded, and the pellet was washed by resuspension in ice-cold Tris/HCl-buffer (1 ml), centrifugation and careful removal of the supernatant. Afterwards, the pellet was resuspended in de-ionized water (500 μl). Twelve replicates of this suspension were combined and concentrated nitric acid was added (550 μl, 65%) to fully digest the cellular material (48 h, 110 °C, pressure vials). The resulting clear solutions were diluted to a final volume of 10 ml with de-ionized water (containing 1 p.p.m. yttrium as internal standard) and subjected to ICP-OES quantification. The obtained p.p.b.-values (1 p.p.b. = 1 μg l−1) for ruthenium were transformed into a molar concentration and the average ruthenium atom count per cell was calculated from the OD of the cultures before decomposition assuming an OD -to-cell-number correlation for E. coli in complex medium of 7.8 × 108 ml−1 OD −1, as described in ref. 34. The cellular metathesis activity was quantified using a fluorescent assay. For this purpose, cell cultures from 96-deepwell plate cultivations (see above) were pelleted by centrifugation (3,220 r.c.f., 12 min) and resuspended in ice-cold Tris/HCl-buffer (500 μl, 50 mM, pH 7.4) containing biot-Ru (2.1 μM). This buffer was supplemented with NaCl (0.9% (w/v)) to adjust to a physiological NaCl concentration. To allow cellular uptake of biot-Ru, the suspensions were incubated on ice for 30 min, spun down (1,260 r.c.f., 8 min) and the supernatant with its excess of cofactor was discarded. The pellets were resuspended in the reaction buffer (160 μl, 100 mM sodium acetate, pH 4.0, 0.5 M MgCl ) and the reaction was initiated by addition of these cell suspensions (150 μl) to the substrate solution (50 μl reaction buffer containing 40 mM precursor 1 and 20% DMSO), leading to a final substrate concentration of 10 mM and 5% DMSO. As the metathesis product umbelliferone 2 is fluorescent, the reaction progress was monitored by fluorescence in a microtiter plate reader (excitation wavelength, 322 ± 4.5 nm; emission wavelength, 440 ± 10 nm; Infinite M1000 PRO, Tecan Group AG) at 37 °C and agitation (6-mm amplitude, orbital). Cell-specific metathesis activity is specified as the slope of the increasing fluorescence signal in the linear range of the reaction normalized by the OD of the respective culture. To generate diversity in the scaffold protein SAV, a focused, semi-rational strategy was pursued: the 20 amino acid residues closest to the ruthenium ion (see Supplementary Table 4) in a related artificial metalloenzyme structure8 were selected and individually randomized in SAVperi by site-saturation mutagenesis using NNK codons30. Degeneration was introduced by application of the Quikchange Site Directed Mutagenesis Protocol (Stratagene) using degenerate oligos (see Supplementary Table 4) and, after transformation, the libraries were checked for diversity by Sanger-sequencing of at least four individual clones before screening. To evaluate the performance of the SAVperi variants, the aforementioned fluorescent metathesis assay was carried out with at least 90 clones from the library in one 96-well plate (three replicates of strains producing ‘wild-type’ SAV (parent) and three replicates of strains carrying an empty vector control (pET-30b(+ ), lacking the gene for SAV ). Evaluating 90 members of an NNK library ensures a >94% likelihood of screening all 20 amino acid residues in the respective position29. To compensate for biological variance, promising clones were isolated and subjected to a replicate assay that was identical to the protocol described above, but using eight independent cultures per clone. After this first screening round, promising residues were ordered according to their potential impact on catalysis and mutations were combined using iterative saturation mutagenesis (ISM)30. To perform kinetic experiments on the artificial metathase, the initially used T7-tagged SAV as well as the quintuple mutant SAVmut that was isolated after ISM were cloned into a cytoplasmic expression vector (see Methods section ‘Cloning of SAV expression constructs’) and purified on an iminobiotin sepharose column as described elsewhere35. The biotin binding capacity was determined using a fluorescent quenching assay36. Kinetic measurements were performed in reaction buffer (200 μl total volume, 100 mM sodium acetate, pH 4.0, 0.5 M MgCl , 11.5% (v/v) DMSO) containing biot-Ru (50 μM) both in the presence and absence of purified SAV (100 μM binding sites of either T7-tagged SAV or SAVmut) as well as the substrate 1 (variable concentrations, 0–5 mM) for fluorescent RCM. The reaction was monitored in a microtiter plate reader as described in Methods section ‘Fluorescent metathesis assay with whole cells’. The maximum velocity of the reaction was determined from the fluorescent signal curve by linear regression. To retrieve kinetic parameters, the reaction velocities were plotted over the respective substrate concentrations using the software GraphPad Prism (GraphPad Software, version 6.05) and applying the integrated ‘Michaelis–Menten’ least-squares fit with no constraints for the maximum velocity V and substrate affinity K . Crystals of SAV and variant SAVmut were obtained at 20 °C within two days by the sitting-drop vapour diffusion technique mixing 1 μl crystallization buffer (1.5 M ammonium sulfate, 0.1 M sodium acetate, pH 4.0) and 4 μl protein solution (26 mg ml−1 lyophilized protein in water). The droplet was equilibrated against a reservoir solution of 100 μl crystallization buffer. Subsequently, single crystals were soaked for two days at 20 °C in a soaking buffer, which was prepared by mixing 1 μl of a 10 mM stock solution of complex biot-Ru (in 50% aqueous DMSO) and 9 μl crystallization buffer. After the soaking, crystals were transferred for 30 s into a cryo-protectant solution consisting of 25% (v/v) glycerol in crystallization buffer. Next, crystals were shock-frozen in liquid nitrogen. Additional soaking of the above metathase crystals with substrate surrogate 1 did not lead to fluorescence. We therefore conclude that the multiple catalytic steps (for example, ligand displacement and cross-metathesis) required to ultimately liberate umbelliferone 2 cannot take place within a crystal. X-ray diffraction data were collected at the Swiss Light Source beam line X06DA at a wavelength of 1 Å and processed with the software XDS37 and AIMLESS (CCP4 Suite)38. The structure was solved by molecular replacement using the program PHASER (CCP4 Suite)38 and the structure 2QCB from the PDB as an input model with ligand and water molecules removed. For structure refinement REFMAC5 (CCP4 Suite)39 and PHENIX.REFINE40 were used. Ligand manipulation was carried out with the program REEL using the small-molecule crystal structure ABEJUM from the Cambridge Structural Database as an input model40. For water picking and electron density and structure visualization, the software COOT41 was used. Figures were drawn with PyMOL (the PyMOL Molecular Graphics System, version 22.214.171.124, Schrödinger, LLC). Crystallographic details, processing and refinement statistics are given in Supplementary Table 5. There is one SAVmut monomer in the asymmetric unit from which a tetramer can be generated by application of two orthogonal crystallographic two-fold symmetry axes. The 12 N-terminal residues of the T7-tag and 25 residues at the C terminus are not resolved, probably owing to disorder. Residual electron density in the biotin-binding site and the biotin vestibule as well as two strong anomalous dispersion density peaks in the biotin vestibule (Extended Data Fig. 3) suggested modelling of complex biot-Ru in two conformations I and II (56% and 44% occupancy, respectively). This projects the ruthenium atom in either one of the two densities, in close proximity to a crystallographic two-fold symmetry axis (Fig. 3b, Extended Data Figs 3, 4c, d). Only partial or no electron density was present for the mesityl linker and the terminal mesityl group, probably owing to high flexibility. In conformer I, the lengthy dimesitylimidazolidine (DMI)-Ru head group reaches into the neighbouring cis-related SAVmut monomer. The I conformer is stabilized mostly by hydrophobic interactions between the distal face of the DMI ligand and amino acid side chains within two neighbouring cis-related 7,8-loops (L1101, S1121, T114Q1, K121R1, L1241, S1122, W1202, K121R2, L1242; superscripts refer to monomers 1 or 2 of the tetrameric SAVmut; Extended Data Fig. 4c). Besides the DMI ligand, two chloride ions could be modelled binding to the ruthenium, but no density was found for the alkylidene, presumably owing to high flexibility and/or low occupancy. The orientation of the chlorides was very similar to that in a small-molecule crystal structure of the Grubbs–Hoyveda second-generation catalyst (ABEJUM from the Cambridge Structural Database), placing them nearly in trans position to each other. The ruthenium is largely solvent-exposed, which could facilitate substrate binding and product release. In Fig. 3b, the alkylidene was modelled binding to the ruthenium (magenta stick model) to highlight its orientation in the biotin vestibule. Conformer II is different from I by a rotation (about 60°) of the DMI-Ru moiety around an axis parallel to the cylinder axis of the SAVmut β-barrel (Fig. 3b, Extended Data Fig. 4d). This rotation places the hydrophobic distal side of the DMI-ligand in proximity to amino acid side chains within loop-5,61 (A86, H87), loop-7,81 (S112, T114Q) and loop-4,53 (D67, S69, A65) of a neighbouring trans-related SAV monomer. Atom N49K-Nε is located in close proximity to ruthenium (2.4 Å). No electron density was found to model chloride ions and the alkylidene ligand bound to the ruthenium. Because the cofactor is bound in close proximity to a two-fold crystal symmetry axis, formation of a cis-symmetry-related neighbouring cofactor by application of the crystal symmetry operation results in extensive steric clashes between the two cofactors in the orientation I. In contrast, coexistence of cofactor pairs I–I or II–II orientation is sterically accessible (Extended Data Fig. 3a, c, e). Normalized B factors of residues within cofactor-flanking loop-7,8 are increased by up to about 0.5 when compared to related SAV structures that crystalized in the same space group and in a very similar unit cell (Extended Data Fig. 5b). This suggests increased loop-7,8 flexibility (Fig. 3b, Extended Data Fig. 4c, d). This flexibility is likely to be caused by three factors: (i) mutation T114Q cleaves a hydrogen-bond between threonine-OγH and T115-carbonyl oxygen (green dashed line in Fig. 3b and Extended Data Fig. 4a, b) and leads to a new hydrogen-bond between T114Q-glutamine-Nε and S112-OγH (red dashed line in Fig. 3b and Extended Data Fig. 4c, d); (ii) mutation A119G in the loop leads to increased entropy; and (iii) mutation V47A reduces steric hindrance between V47A in loop-3,42 and W120 in loop-7,81 (Fig. 3b and Extended Data Fig. 4c, d). The overall structure of complex biot-Ru–SAV is virtually identical to that of complex biot-Ru–SAVmut (root-mean-square deviation, r.m.s.d. = 0.25 Å). As in the mutant, two strong residual electron density peaks (F − F cofactor omit map: 12σ (conformer I) and 11σ (conformer II)) and two anomalous dispersion density peaks (9σ (conformer I) and 5σ (conformer II)) were located within the biotin vestibule of a SAV monomer at the interface between two symmetry-related SAV monomers (Extended Data Fig. 3b, d, f). The same two cofactor conformations I and II found in mutant SAVmut were modelled in the biotin binding vestibule of SAV with an occupancy of 50% for each conformer I and II (Extended Data Fig. 4a, b). The side chain of residue L110 adopts two conformations with 50% occupancy each (Extended Data Fig. 4a, b). The close proximity of the terminal methyl group in L110 conformation A to the aromatic mesityl ring (distance between L110-CδH and mesityl of 4.4 Å) of cofactor conformation I suggests a stabilizing σ–π interactions (Extended Data Fig. 4a, red star). The L110 side chain conformation B can coexist only with cofactor conformation II (Extended Data Fig. 4b). This hypothesis is supported by the fact that the same L110 side chain conformation A is found in complex [(Cp*)Ir(Biot-p-L)Cl]-SAV-S112A (PDB, 3PK2), which has an aromatic ring located in the same position as the mesityl linker in structure biot-Ru–SAV, suggesting a similar σ–π interaction. In contrast, the side chain of L110 in apo-SAV (PDB, 2BC3) adopts conformation B. In complex biot-Ru–SAV, a water molecule is bound in proximity to L110 with 50% occupancy (Extended Data Fig. 4b). Steric clashes with L110 side chain in conformation A and the NHC ligand of biot-Ru conformer I suggest that the water is present only with L110 conformation B and biot-Ru conformer II. Additionally, the side chain of L124 adopts two conformations, each with 50% occupancy. Only conformation L124A does not undergo steric clashes with a methyl group of the bridging mesityl moiety of cofactor conformer I (Extended Data Fig. 4a, b). Together, the conformational side chain flexibility of residues L110 and L124 reflects the presence of the two cofactor conformations I and II. In contrast to artificial metalloenzyme biot-Ru–SAVmut, the normalized B factors of residues within the cofactor-flanking loop-7,8 in complex biot-Ru–SAV do not show increased values when compared to those in related crystal structures (Extended Data Fig. 5b). Indeed, a hydrogen-bond is formed between T114-OγH and T115-carbonyl oxygen in complex biot-Ru–SAV that could rigidify the loop (Extended Data Fig. 4a, b). The conversion for the product 4 was quantified by 1H-NMR. For this purpose, a deuterated reaction buffer was prepared from acetic acid-d , dry MgCl and D O with the same concentrations as for the reaction buffer used for substrate 1 (100 mM acetate, 0.5 M MgCl ). The pH was adjusted to 3.6 by addition of 1 M NaOD in D O (with respect to pD = pH + 0.4). For the reaction, 300 μl of a substrate 3 stock solution (100 mM in deuterated reaction buffer) was mixed with 291 μl of either a solution of SAV, SAVmut, or SAVmut2 (200 μM binding sites in deuterated reaction buffer) or plain deuterated reaction buffer (for samples without SAV or any of the SAV variants). Afterwards, 9 μl of a biot-Ru (or HGII/AQM) stock solution (3.34 mM in DMSO-d ) was added to obtain a final concentration of 50 μM and the reaction was performed for 16 h at 37 °C and 200 r.p.m. The mixture was analysed by 1H-NMR and the yield of the reaction product 4 was quantified by comparing integrals (I) of the product 4 peaks at 3.41 p.p.m. and 2.05 p.p.m. and the substrate 3 peaks at 3.33 p.p.m. and 1.91 p.p.m. using: yield = I /(I + I ). To quantify the conversion of substrate 5, a 97 μl aliquot of either SAV solution (200 μM SAV binding sites in reaction buffer) or plain reaction buffer (for samples without SAV variants) was mixed with 100 μl of a stock solution of substrate 5 (20 mM in reaction buffer). Subsequently, 3 μl of the respective catalyst/cofactor stock solution (3.34 mM in DMSO) was added to obtain a final concentration of 50 μM. The reaction was performed for 16 h at 37 °C and 200 r.p.m. Then, an aqueous solution of benzyltriethylammonium chloride (100 μl, 10 mM) was added as an internal standard and 700 μl of methanol was added. The mixture was cleared by centrifugation and 250 μl of the supernatant was mixed with 750 μl of water for the final quantification of product 6 by UPLC-MS. For the kinetic experiment, the reaction mixture was scaled up to a total volume of 1 ml and 50 μl aliquots of this mixture were collected at different time points and immediately injected into 950 μl of a quenching solution (0.5 mM potassium cyanoacetate, 0.25 mM benzyltriethyl-ammonium chloride (internal standard) in 50% aqueous methanol). After removal of precipitated protein by centrifugation, the supernatant was analysed by UPLC-MS. To quantify the cellular metathesis activity for substrate 5, a protocol analogous to that applied for the umbelliferone precursor 1 was applied. The substrate 5 (final concentration 10 mM) was added to whole cells and the samples were incubated at 37 °C and 300 r.p.m. for 16 h. To quantify the conversion for the non-fluorescent product 6, an extraction was performed: 800 μl of methanol was added to each sample and an extraction was carried out (one hour with vigorous shaking, 800 r.p.m. at room temperature). The samples were cleared by centrifugation, the supernatant was diluted with water (factor four) and analysed by UPLC-MS using a calibration curve recorded for product 6. No statistical methods were used to predetermine sample size.
News Article | November 7, 2016
Eleven Presentations Evaluating Marketed and Investigational Compounds for Hepatic Veno-Occlusive Disease (VOD) and Acute Myeloid Leukemia (AML) Presentations Include a Sub-Analysis of Phase 3 Data for Vyxeos (CPX-351), an investigational product for the treatment of AML Patients DUBLIN, Nov. 7, 2016 /PRNewswire/ -- Jazz Pharmaceuticals plc (Nasdaq: JAZZ) announced today that eleven abstracts, including four oral presentations, supporting the company's hematology and oncology portfolio will be presented at the 58th American Society of Hematology (ASH) Annual Meeting and Exposition in San Diego, California, December 3-6, 2016. "The data presentations at ASH reflect our efforts in advancing our diversified pipeline of programs in hematology and oncology, including rare blood disorders such as acute lymphoblastic leukemia (ALL) and AML, and in complications of hematopoietic stem-cell transplantation (HSCT) such as hepatic VOD," said Karen Smith, M.D., Ph.D., global head of research and development and chief medical officer at Jazz Pharmaceuticals. "Of note, we look forward to sharing a post-hoc sub-analysis of Phase 3 survival data following allogeneic HSCT in older high-risk AML patients that compares CPX-351, also known as Vyxeos, with the standard of care." The following oral and poster presentations focusing on Defitelio® (defibrotide sodium) injection, Erwinaze® (asparaginase Erwinia chrysanthemi) and CPX-351(cytarabine and daunorubicin liposome injection) will be presented at ASH. Additionally, one Jazz-sponsored Investigator Initiated Research poster presentation focusing on CPX-351 as an investigational agent for the treatment of AML will be presented at ASH. Full details of the ASH 2016 annual meeting can be found here (http://www.hematology.org/Annual-Meeting/) and abstracts can be found here (https://ash.confex.com/ash/2016/webprogram/start.html). About Defitelio1 In the U.S., Defitelio® (defibrotide sodium) injection 80mg/mL received FDA marketing approval on March 30, 2016 for the treatment of adult and pediatric patients with hepatic veno-occlusive disease (VOD), also known as sinusoidal obstruction syndrome (SOS), with renal or pulmonary dysfunction following hematopoietic stem-cell transplantation (HSCT) and is the first and only FDA-approved therapy for patients with this rare, potentially fatal complication. Defitelio is contraindicated in patients currently taking anticoagulants or fibrinolytics and in patients who are allergic to Defitelio or any of its ingredients. Defitelio may increase the risk of bleeding and should be withheld or stopped if significant bleeding occurs. Patients should be monitored for allergic reactions, especially if there is a history of previous exposure to Defitelio. The most common side effects of Defitelio are decreased blood pressure, diarrhea, vomiting, nausea and nose bleeds. Please see full for Defitelio. (https://defitelio.com/DefitelioPI.pdf) In Europe, defibrotide is marketed under the name Defitelio®▼(defibrotide). In October 2013, the European Commission granted marketing authorization to Defitelio under exceptional circumstances for the treatment of severe VOD in patients undergoing HSCT therapy. It is the first and only approved treatment in Europe for severe VOD. In Europe, Defitelio is indicated in patients over one month of age. It is not indicated in patients with hypersensitivity to defibrotide or any of its excipients or with concomitant use of thrombolytic therapy. ▼This medicinal product is subject to additional monitoring. This will allow quick identification of new safety information. Healthcare professionals are asked to report any suspected adverse reactions via the national reporting system found under section 4.8 of the SmPC. (http://www.ema.europa.eu/ema/index.jsp?curl=/pages/medicines/human/medicines/002393/human_med_001646.jsp) About VOD HSCT is an aggressive, potentially curative procedure to treat patients with malignant and non-cancerous hematologic disorders such as leukemia, lymphoma and aplastic anemia, and congenital immunodeficiency and autoimmune disorders.2 VOD is a rare complication of HSCT, which occurs in approximately 9-14% of HSCT patients.3,4 Hepatic VOD, also known as SOS, is an early and life-threatening complication affecting the sinusoidal endothelial cells of the liver, which can typically occur within the first 21 days following HSCT.4,5 Hepatic VOD progresses to multi-organ dysfunction in approximately 30-50% of cases.5 VOD with multi-organ dysfunction (MOD) is associated with an overall mortality (death) rate of 84%.3 MOD is characterized by the presence of renal or pulmonary dysfunction.6,7 VOD is often characterized by sudden weight gain, hepatomegaly (abnormally enlarged liver), and elevated bilirubin.6,7 About Erwinaze Erwinaze® (asparaginase Erwinia chrysanthemi) is currently approved in the U.S. for administration via intramuscular injection or via intravenous infusion in conjunction with chemotherapy. It is indicated as a component of a multi-agent chemotherapeutic regimen for the treatment of patients with acute lymphoblastic leukemia (ALL) who have developed hypersensitivity to E. coli-derived asparaginase.8 Erwinaze is derived from the bacterium Erwinia chrysanthemi and is therefore immunologically distinct from E. coli-derived asparaginase and suitable for patients with hypersensitivity to E. coli-derived treatments9. Outside of the U.S., Erwinaze is sold under the name Erwinase®. Please consult local labeling for product information specific to your country. Erwinaze is contraindicated in patients who have had serious allergic reactions to Erwinaze, or had serious swelling of the pancreas, serious blood clots, or serious bleeding with past L-asparaginase treatment. Erwinaze should be discontinued if any of the following occur: serious allergic reactions, including a feeling of tightness in the throat, unusual swelling/redness in the throat and/or tongue, or trouble bleeding; or severe inflammation of the pancreas. Glucose intolerance has been reported, which in some cases may be irreversible. If blood clots of bleeding occur, discontinue Erwinaze until symptoms resolve. The most common side effects of Erwinaze are allergic reactions, too much sugar in the blood, fever, swelling of the pancreas, local reactions (swelling, rash, etc. where the needle entered the skin), vomiting, nausea, blood clots, liver problems, stomach pain/discomfort, and diarrhea. Please see full Prescribing Information for Erwinaze. (https://www.jazzpharma.com/wp-content/uploads/2016/01/erwinaze-en-PI.pdf) About Vyxeos (CPX-351) CPX-351 (cytarabine and daunorubicin liposome injection) is an investigational product being evaluated for the treatment of AML and is a combination of cytarabine and daunorubicin encapsulated within a nano-scale liposome at a 5:1 molar ratio. The proposed trade name, Vyxeos™, is conditionally approved by the FDA and is subject to confirmation upon approval of the NDA. CPX-351 was granted orphan drug status by the FDA and the European Commission for the treatment of acute myeloid leukemia. CPX-351 was granted Breakthrough Therapy Designation for the treatment of adults with therapy-related AML or AML with myelodysplasia-related changes and was also granted Fast Track designation by the FDA for the treatment of older patients with secondary AML. On October 3, 2016 Jazz announced the initiation of a rolling submission of a New Drug Application (NDA) to the FDA, seeking marketing approval of CPX-351 for the treatment of AML. About Acute Myeloid Leukemia Acute myeloid leukemia (AML) is a rapidly progressing and life-threatening blood cancer that rises in frequency with age.16 The American Cancer Society estimates that there will be 19,950 new cases of AML and 10,430 deaths from AML in the U.S. in 2016.10 In the European Union, the number of new cases is estimated to be 20,100 in 2016.11 The median age at diagnosis is 67 and with rising age there is progressive worsening of prognosis.10,12 Advancing age is associated with increasing risk of specific chromosomal/mutational changes and risk of pre-malignant marrow disorders which give rise to more aggressive and less responsive forms of AML.13,14 As patients age there is also reduced tolerance for intensive chemotherapy.15 As a consequence, advances in supportive care, intensive chemotherapy, and bone marrow transplantation have primarily benefitted younger patients with approximately one third of patients 18-60 years of age achieving cure.13,15 Older patients have not achieved higher rates of cure or improved upon a 5-year survival rate of 10-20% in spite of 40 years of research.15,16 About Jazz Pharmaceuticals Jazz Pharmaceuticals plc (Nasdaq: JAZZ) is an international biopharmaceutical company focused on improving patients' lives by identifying, developing and commercializing meaningful products that address unmet medical needs. The company has a diverse portfolio of products and product candidates, with a focus in the areas of sleep and hematology/oncology. In these areas, Jazz Pharmaceuticals markets Xyrem® (sodium oxybate) oral solution, Erwinaze® (asparaginase Erwinia chrysanthemi) and Defitelio® (defibrotide sodium) in the U.S. and markets Erwinase® and Defitelio® (defibrotide) in countries outside the U.S. For more information, please visit www.jazzpharmaceuticals.com. References: 1 Defitelio (defibrotide sodium) [package insert]. Palo Alto, CA: Jazz Pharmaceuticals; March 30, 2016. 2 Ikehara S. New strategies for BMT and organ transplantation. Int J Hematol. 2002;76(Suppl 1):161-4. 3 Coppell JA, Richardson PG, Soiffer R, et al. Hepatic veno-occlusive disease following stem cell transplantation: incidence, clinical course, and outcome. Biol Blood Marrow Transplant. 2010;16(2):157-168. 4 Tsirigotis PD, Resnick IB, Avni B, et al. Incidence and risk factors for moderate-to-severe veno-occlusive disease of the liver after allogeneic stem cell transplantation using a reduced intensity conditioning regimen. Bone Marrow Transplant. 2014;49(11):1389-1392. 5 Carreras E, Díaz-Beyá M, Rosiñol L, et al. The incidence of veno-occlusive disease following allogeneic hematopoietic stem cell transplantation has diminished and the outcome improved over the last decade. Biol Blood MarrowTransplant. 2011;17(11):1713-1720. 6 Carreras E. How I manage sinusoidal obstruction syndrome after haematopoietic cell transplantation. Brit J Haematol. 2015 Feb.; 168 (4); 481-91. 7 Mohty M, Malard F, Abecassis M, et al. Sinusoidal obstruction syndrome/veno‐occlusive disease: current situation and perspectives—a position statement from the European Society for Blood and Marrow Transplantation (EBMT). Bone Marrow Transplant. 2015;50(6):781‐789. 8 Erwinaze® (asparaginase Erwinia chrysanthemi) [prescribing information]. Palo Alto, CA: Jazz Pharmaceuticals, Inc., December 2014. 9 Pieters R, Hunger SP, Boos J, et al. L-asparaginase treatment in acute lymphoblastic leukemia: a focus on Erwinia asparaginase. Cancer. 2011 Jan 15; 117(2): 238–249. 10 American Cancer Society. Leukemia--Acute Myeloid (Myelogenous). Detailed Guide. What are the key statistics about acute myeloid leukemia? http://www.cancer.org/cancer/leukemia-acutemyeloidaml/detailedguide/leukemia-acute-myeloid-myelogenous-key-statistics. Last revised February 22, 2016. Accessed November 3, 2016. 11 Decisions Research Group. Acute Myeloid Leukemia. https://decisionresourcesgroup.com/report/141439-biopharma-acute-myeloid-leukemia/. Published November 2015. Accessed November 3, 2016. 12 Baer M, George S, Sanford B, et al. Escalation of daunorubicin and addition of etoposide in the ADE regimen in acute myeloid leukemia patients aged 60 years and older: Cancer and Leukemia Group B Study 9720. Leukemia. 2011;25(5):10.1038/leu.2011.9. doi:10.1038/leu.2011.9. 13 Ferrara F, Schiffer CA. Acute myeloid leukaemia in adults. Lancet. 2013 Feb 9;381(9865):484-495. 14 Dohner H, Estey EH, Amadori S, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood. 2010 Jan 21;115(3):453-474. 15 Stone RM, O'Donnell MR, Sekeres MA. Acute myeloid leukemia. Hematology Am Soc Hematol Educ Program. 2004:98-117. 16 Kadia TM, Ravandi F, Cortes J, Kantarjian H. New drugs in acute myeloid leukemia. Ann Oncol. 2016 May;27(5):770-778.
Sun G.,Wuhan University |
Sun G.,Hei Longjiang Institute of Geological Survey |
Sun G.,Northeast Petroleum University |
Li Z.,Institute of Geochemistry Chinese Academy of science |
And 4 more authors.
Atmospheric Environment | Year: 2013
Mercury (Hg) contamination in urban area is a hot issue in environmental research. In this study, the distribution, sources and health risk of Hg in dust from 69 kindergartens in Wuhan, China, were investigated. In comparison with most other cities, the concentrations of total mercury (THg) and methylmercury (MeHg) were significantly elevated, ranging from 0.15 to 10.59mgkg-1 and from 0.64 to 3.88μgkg-1, respectively. Among the five different urban areas, the educational area had the highest concentrations of THg and MeHg. The GIS mapping was used to identify the hot-spot areas and assess the potential pollution sources of Hg. The emissions of coal-power plants and coking plants were the main sources of THg in the dust, whereas the contributions of municipal solid waste (MSW) landfills and iron and steel smelting related industries were not significant. However, the emission of MSW landfills was considered to be an important source of MeHg in the studied area. The result of health risk assessment indicated that there was a high adverse health effect of the kindergarten dust in terms of Hg contamination on the children living in the educational area (Hazard index (HI)=6.89). © 2013 Elsevier Ltd.
News Article | December 5, 2016
HONG KONG, 5 décembre 2016 /PRNewswire/ -- L'InterContinental Grand Stanford Hong Kong a retenu l'attention du gotha mondial du tourisme lors du grand gala de clôture des World Travel Awards (WTA) 2016, qui se tenait aux Maldives, remportant leu...
News Article | December 8, 2016
Liviu Dragnea, leader of the Social Democratic Party of Romania (PSD), today outlined a series of budget proposals designed to raise revenues but leave more in the pockets of ordinary Romanians. The budget unveiled by Mr. Dragnea puts into concrete terms the electoral program that has spearheaded the PSD campaign of the past several months, focusing on a robust set of pro-growth fiscal measures and branded under the slogan "Dare to Believe in Romania." Mr. Dragnea also announced a major increase in defence spending, in keeping with commitments by Nato members to spend at least 2% of GDP on military preparedness, to join just five other member states currently meeting that target. This would be a significant policy change for Romania, representing a 40% increase in expenditure. Despite continued economic growth arising from the pre-2016 PSD government's programs, national revenues under the current caretaker government have actually declined, the first time this has occurred since 2009. In recent weeks, in the run-up to parliamentary elections this Sunday (Dec. 11), Mr. Dragnea has made a series of announcements intended to convert the country's growth into genuine benefits for Romanian citizens. "This budget shows with clear figures that our proposals will put the economic growth into people's pockets, through higher wages and pensions as well as investments in education, health and infrastructure, so that Romanians can enjoy a better life as part of the growing middle class," Mr. Dragnea told a Bucharest press conference today. He said the next PSD government's priorities would be higher incomes for Romanians, investments in education, health and infrastructure and lower taxes. The plan anticipates budgetary revenues of 254 billion lei (approximately EUR 56.5 billion at current rates), an increase of about 10% from the current year. Main sources would be VAT, excise taxes, other service charges and income tax revenues. In addition, funds from the European Union are forecast to increase to 21 billion lei in 2017. The PSD has been highly critical of the current government's failures to draw down funds from the EU having failed to meet administrative requirements. Mr. Dragnea proposed budget cuts for the ministries of finance, energy and communications, a 20.9% cut in the budget of the Senate and 17.9% cut in the budget of the Chamber of Deputies. "Our priorities will be to increase spending on health, education, wages, pensions, agriculture and defense while cutting wasteful spending in Parliament administration and the government ministries," he said. By setting defence spending at 2% of GDP, Romania will be joining the US, Greece, Poland, Estonia and the UK as the only NATO members currently meeting or exceeding member countries' commitments, agreed to at NATO's 2014 summit in Wales. "As a strategically located state, we have a responsibility to meet our obligations to our defence partners and to invest in our own security," Mr. Dragnea said. In several pre-election announcements, Mr. Dragnea has already announced the main elements of the PSD economic plan. These include a major investment program, dramatic tax changes, steps to attract desperately needed medical professional back to Romania and a program to give bootstrap support for new businesses. A national reindustrialization program is designed ultimately to generate 45,000 new jobs. Linchpin of the plan is a EUR 10 billion (45 billion Romanian Leu) state investment fund, the Sovereign Fund for Development and Investment or SFDI (Fondul Suveran de Dezvoltare si Investitii, or FSDI), which is to be created from the assets owned by the state in 200 companies. The fund is expected to generate revenues of over 50 billion leu in the next four years, as dividends from those companies are reinvested, and private investments are attracted. About the Social Democratic Party of Romania The PSD, in Romanian Partidul Social Democrat, was originally formed in 1992 as a party of the center-left and is currently the largest grouping in both lower and upper houses of the nation's parliament, while also controlling more than half of the mayoralties and over 65% of local and county councils, including the capital of Bucharest. The PSD paved the way for Romania's historic accession process into the EU in 2007, and today holds 16 of the country's 32 MEPs. Its 2012-15 government was considered one of Romania's best, leading the country's emergence from the economic crisis and achieving rapid growth, together with a sharp increase of people's living standards. Liviu Dragnea, the PSD's current president, was elected party leader in 2015 and has since led a series of reforms that have positioned the PSD to form Romania's next government. Mr. Dragnea is a former Deputy Prime Minister and Minister for Regional Development. For further information: Steluta Negoita, +40 730 650 545, email@example.com in Bucharest or Zhenya Harrison, +44 (0)20 3397 2825 or firstname.lastname@example.org in London.
Liu Z.-H.,LEU |
Wang F.,LEU |
Construction and Building Materials | Year: 2016
SiO2 aerogel is a solid material with three-dimensional network structure, has a very low density and low thermal conductivity, can be used as a kind of high efficient heat preservation and heat insulation material, as well as SiO2 aerogel particles. In this work, we have successfully prepared a mortar with SiO2 aerogel particles. Density, mechanical properties, softening coefficient, autogenous shrinkage, antifreeze performance and thermal conductivity properties were studied when the SiO2 aerogel particles replacement ratio was 10%, 20%, 30%, 40%, 50% and 60%. The mortar based on SiO2 aerogel particles shows a density of ∼1.2 g/cm3, a compressive strength and flexural tensile strength of ∼2.15 MPa and ∼0.45 MPa, a thermal conductivity of ∼0.1524 W/m·K at the aerogel content of 60 vol.%. And on this basis, the thermal conductivity was focused on study when fiber, air-entraining agent and powder were added into the mortar. The results show that when the dosage was 0.2%, 0.05% and 1% respectively, the thermal conductivity was the lowest (0.0859 W/m·K). © 2016 Elsevier Ltd
Deng Z.,LEU |
Cheng H.,LEU |
Wang Z.,LEU |
Zhu G.,LEU |
Construction and Building Materials | Year: 2016
The compressive behavior of the cellular concrete utilizing millimeter-size spherical saturated SAP with different porosities was experimentally investigated under quasi-static and high strain-rate loadings. Quasi-static tests at strain rate of 10−5/s were conducted using a servo-hydraulic material test system. Impacting tests at strain rates ranging from 70/s to 140/s were performed using Φ74 mm conic variable cross-sectional split Hopkinson pressure bar (SHPB) equipment. The strain rate effects on the failure pattern, compressive strength, elastic modulus and critical strain were studied. The results indicated that the compressive strength and elastic modulus increased with increasing strain rate and displayed clear strain-rate dependence. At high strain rates the compressive strength decreased with increasing the porosity, while dynamic increase factor of the compressive strength (DIF-fc) showed a reverse tendency. Furthermore, the elastic modulus and its dynamic increase factor (DIF-E) both decreased with increasing the porosity at given high strain rate. © 2016 Elsevier Ltd
Yi B.,LEU |
Mi H.,LEU |
Guangxue Jishu/Optical Technique | Year: 2012
A new method of DSC's imaging luminance measurement based on HDR image processing is proposed. The time scale exposure series of LDR images to compose a high dynamic range image are used in the method. Then the tristimulus value is calculated from the RGB output by using the color space transformation. Finally the estimated value of luminance is computed by using the calibration coefficient of the digital camera optic-electric conversing function. The luminance measurement results in a exterior test experiment have shown the approach's effectiveness.
Yantu Gongcheng Xuebao/Chinese Journal of Geotechnical Engineering | Year: 2014
Thorough researches on unsaturated soils and special soils including fill, collapsible loess, expansive soil, bentonite and sandy clay, are carried out, and a series of novel results are obtained. A lot of instruments and equipments suitable for unsaturated soils and special soils, such as oedometer, direct shear apparatus, gas permeation device, multifunction triaxial apparatus and temperature-controlled triaxial apparatus as well as CT-triaxial apparatus, are successfully developed. Important mechanical properties and laws of water flow, air flow, deformation, strength, yield, moisture and meso-structure of unsaturated soils and special soils are revealed. An axiomatics theoretical system of geomechanics and the stress state variables of unsaturated soils are established. The formulae for effective stresses isotropic unsaturated soils and anisotropic porous media are proposed. The constitutive hierarchy including nonlinear model, elasto-plastic model, coupled temperature-mechanical model and meso-structure damage model as well as the general model for soil-water characteristics curve are established. The 3-D consolidation theory based on the theory of mixture and the hierarchy of consolidation model are created, the relevant software to solve 2-D consolidation problems is programmed independently, and the analytical solutions to 1-D problems and the numerical solutions to 2-D problems are obtained. Many difficult problems such as deformation and stability of dams and embankments are solved using the above achievements. It is indicated that the proposed theories can be adopted to guide engineering practices and to be as the theoretical basis for engineering decision.
Leu | Date: 2015-05-20
A recognition device of a foot action includes a sensing module, a processing module, and a display module. The sensing module senses the foot action to generate at least one sensing signal corresponding to the foot action, and a form of the sensing signal is a waveform. The processing module is electrically connected the sensing module. The processing module recognizes an action mode corresponding to the foot action according to the waveform and converts the waveform into input data according to the action mode. The display module is electrically connected the processing module and displays the input data. Furthermore, a recognition method of the foot action is also disclosed herein.