Uranium was totally extracted by both ionic liquids over a broad pH range (2–8), while the other radionuclides were removed with differing efficacies depending on the respective pH value.Two novel maltol-based ionic liquids, namely [A336][Mal] and [C101][Mal], were synthesized as potential extracting agents for radionuclides from water. These two room temperature task-specific ionic liquids could be easily prepared by anion metathesis starting from commercially available materials. The isolated compounds were characterized by standard analytical methods. Their application as extraction agent for UThe online version of this article (doi:10.1007/s10967-014-3782-x) contains supplementary material, which is available to authorized users. Previous liquid–liquid extraction strategies for uranium revealed high efficacies in the case of [A336][TS] and [A336][SCN]  values of these ionic liquids were determined. The extraction efficacies of two ionic liquids with regard to the naturally occurring radionuclides 238U and 234U, 234Th, 226Ra, 210Pb and its progenies 210Bi and 210Po were investigated.Herein we report the synthesis of two new TSILs, tricaprylmethylammonium maltolate and tetradecyltrihexylphosphonium maltolate Fig.  for the H)-pyrone  and Cyphos IL 101  were purchased from Sigma Aldrich. Potassium hydroxide  and Aliquat 336® -alkylmethyl, chlorides, >75 %) were obtained from Acros. Utilized solvents were of HPLC grade and used without further purification. 1H and 13C NMR spectra were recorded on a Bruker Avance IIITM spectrometer in DMSO-d6 at 298 K using standard pulse programs at 500.10 (1H) and 125.76 (13C) MHz. UV–Vis spectra were performed on an Agilent 8453 spectrophotometer in the range of 200–1,000 nm in methanol. ATR-IR spectra were measured with a Bruker Vertex 70 Fourier transform IR spectrometer. TOC measurements were performed on a TOC-V CHP analyser (Shimadzu) and density measurements with a calibrated glass pycnometer (Klaus Hofmann). Viscosity measurements were conducted using a Kinexus rheometer . The water content of [A336][Mal] and [C101][Mal] was determined utilizing a Mettler Toledo DL39 Karl Fischer (KF) coulometer; each ionic liquid was measured in triplicates. In-house standard solutions of different radionuclides were used for the extraction experiments. The uranium standard contained 27.7 μg Unat. in 10 μL diluted HNO3, corresponding to an activity of 682 mBq (238U and 234U), as well as 341 mBq 234Th  in radioactive equilibrium with 238U. The 210Pb  standard solution had an activity concentration of 580 mBq/10 μL. The progenies 210Bi  and 210Po  are in radioactive equilibrium with 210Pb, which means that each of them have an activity concentration of 580 mBq/10 μL. For radium extraction we used 2 mL of the reference material IAEA-431 (29.5.2001) containing 50 mBq 226Ra. Ingrown 222Rn left the sample during shaking with the IL for 226Ra extraction; as the activity measurements were always done immediately after the extraction step the in-growth of 222Rn and its short lived progenies was prevented.3-Hydroxy-2-methyl-4 in methanol (5 mL) was added and the reaction mixture was stirred for further 4 h. After complete evaporation, the residue was extracted with ethyl acetate/water (7 mL/4 mL). The separated organic phase was dried over Na2SO4, evaporated under reduced pressure and dried invacuo, yielding an orange viscous oil. Yield: 122 mg (95 %). 1H NMR : δ = 7.71 , 6.04  3.15–3.25 , 2.96 , 2.19 , 1.56–1.66 , 1.20–1.37 , 0.83–0.91 . 13C{1H} NMR : δ 173.2, 155.0, 149.7, 143.6, 114.0, 61.2, 61.0, 48.0, 31.8, 31.6, 29.4, 29.3, 29.2, 28.9, 28.9, 26.3, 26.2, 22.6, 22.5, 21.8, 14.4. IR : 2925, 2857, 1620, 1463, 1377, 1260, 1196, 1073, 916, 848, 661, 628 cm−1. UV–Vis in MeOH, λ, nm : 215 (10690), 275 (8161).[A336][Mal]: To a solution of maltol  in methanol (5 mL) potassium hydroxide  was added in one portion and the solution was stirred for 10 min at room temperature. Aliquat 3362SO4, evaporated under reduced pressure and dried invacuo, yielding an orange viscous oil. Yield: 149 mg (94 %). 1H NMR : δ = 7.91 , 6.21  2.22 , 2.15–2.21, 1.43–1.53 , 1.34–1.43 , 1.21–1.34 , 0.83–0.92 . 13C{1H} NMR : δ 173.0, 155.1, 149.6, 143.4, 116.8, 114.0, 31.8, 30.9, 30.5, 30.4, 30.3, 30.2, 29.5, 29.5, 29.4, 29.2, 29.1, 28.6, 22.6, 22.3, 21.1, 21.0, 18.3, 18.2, 17.9, 17.8, 14.4, 14.3. IR : 3251, 3061, 2923, 2854, 1653, 1629, 1458, 1369, 1255, 1223, 1197, 1077, 1022, 919, 848, 688 cm−1. UV–Vis in MeOH, λ, nm : 215 (16380), 277 (12392).[C101][Mal]: To a solution of maltol  in methanol (5 mL) potassium hydroxide  was added in one portion and the solution was stirred for 10 min at room temperature. Tetradecyltrihexylphosphonium chloride  in methanol (5 mL) was added and the reaction mixture was stirred for further 4 h. After complete evaporation, the residue was extracted with ethyl acetate/water (7 mL/4 mL). The separated organic phase was dried over Nanat., 210Pb, or 226Ra . Samples were shaken overnight at 100 rpm and subsequently centrifuged for 30 min at 3,500 rpm [the relative centrifugal force (RCF) is 1646] for phase separation. The yield of the various extractions was determined by measuring the remaining activity concentration of the respective radionuclides in the aqueous phase by liquid scintillation counting (LSC). The advantage of this method is twofold: an aliquot of the aqueous solution can be measured directly without further sample treatment, and α- and β-emitters present in the same sample can be measured simultaneously. The poor energy resolution in the α-spectrum is of no relevance, as the 234U/238U ratio is not altered by the extraction in any way.Extractions were performed by weighting selected amounts (100–200 mg) of ionic liquids in 15 mL polypropylene tubes and addition of about 10 mL of aqueous solution containing a known amount of UAll extraction experiments were done at least twofold with satisfying agreement. The maximum difference of the extraction yields from an aqueous solution  measured for two different batches of the same IL were 20 %, comprising also the 1σ-counting uncertainties of 5–10 %. These relatively high counting uncertainties are due to the fact that high extraction yields correspond to low activities left in the aqueous phase, which is then measured. By extending the counting time the uncertainties could be reduced.234Th (+ 234Pa), and 210Pb is in radioactive equilibrium with its progenies 210Bi  and 210Po (α-emitter), pulse shape analysis was used to differ between α- and β-counts .For the LSC measurement an aliquot of the aqueous phase  was transferred to a 20 mL vial and mixed with the scintillation cocktail HiSafe III™. After cooling, the activity concentrations of the respective radionuclides were measured with a Quantulus™ 1220 low-level liquid scintillation counter . Counting times were 300 min or less, depending on the activity of the respective samples. As the uranium solution contains also the β-emitter 3  and potentiometric titrated using AgNO3 as titrant based on two independent experiments.Leaching of the ionic liquids into the water phase was tested the following way: 100 mg of IL were shaken with double distilled water (5 mL) for 24 h. 1.2 mL of the solution were diluted with 10.8 mL double distilled water. The samples were acidified to pH 2 with HCl and purged with carrier gas for 5 min prior to combustion in the TOC analyser. The purities of the tested ionic liquids were quantified by their residual chloride ions. The chloride content was determined by dissolving the sample in 10 mL ethanol/water (1:1), acidified with HNO1H NMR. Afterwards, either Aliquat 336® (a mixture of quaternary ammonium compounds) or Cyphos IL101 was added and after aqueous work up the desired products were isolated in high yield (>90 %) and sufficient purity. An excess of maltol was necessary in both syntheses due to the high solubility of maltolate in water. The isolated ionic liquids were characterized by standard analytical methods .The two maltol-based ionic liquids were prepared by anion metathesis, which is a fast and sustainable standard strategy in literature . Therefo1H NMR spectroscopy . The chloride content of the ionic liquids [A336][Mal] and [C101][Mal] has been found to be 1.5 and 1.7 %, respectively, by argentometric titration  is present in different species depending on the pH value of the solution. For example, at pH > 2.5 UO2(OH)+, (UO2)2(OH)22+ and (UO2)3(OH)5+ species are predominant whereas at pH < 2.5 UO22+ is prevalent [234Th is a progeny of 238U and in the case of [C101][Mal] the extraction of thorium decreased at higher pH which could be explained by lower affinity of 234Th(OH)22+ and other hydrolysis products to this IL compared to 234Th4+ present at lower pH values. In contrast, best extraction rate for 234Th with [A336][Mal] could be found at pH 6. 210Pb was removed less than 20 % of either [A336][Mal] or [C101][Mal] in all experiments and the pH value had no significant impact on the elimination. Up to 80 % of 210Po was removed at pH 2–6 with [A336][Mal], whereas [C101][Mal] was slightly less effective at pH > 4. Interestingly, at pH values ≥ 6, the extraction rate for 210Po decreased significantly for both compounds. Similar extraction data was observed for the 210Bi(III) removal of [A336][Mal] and [C101][Mal] with a maximum of about 40 % at pH 8. 226Ra was not extracted by both maltol-derived ionic liquids. At pH values higher than 6, only uranium could be completely eliminated from the radioactive solution.Due to the known high affinity of the maltolate to a broad range of different metal ions, extraction experiments with a series of radionuclides were performed to investigate potential applications. It was found that uranium was quantitatively extracted by [A336][Mal] and [C101][Mal] Fig.  and the revalent . Howeverrevalent . 234Th i210Po and 234Th were well extracted; whereas no or only low removal of either 226Ra, 210Bi or 210Pb was observed. Back extraction experiments with different stripping agents are ongoing.Herein, we report the straight-forward and cost-saving synthesis of two new maltol-based ionic liquids with tricaprylmethylammonium and tetradecyltrihexylphosphonium as counterions from commercially available precursors by anion metathesis. The obtained compounds were characterized by standard analytical methods and the physicochemical properties were determined. Although a high leaching of the maltol-based ionic liquids was observed, the extraction experiments with different radionuclides were very promising. The extraction of uranium showed a quantitative efficiency for [A336][Mal] and [C101][Mal] over the investigated pH range (2–8), therefore, this method represents a suitable way for uranium removal. Additionally, Overall, these investigations are the first step for the development of maltol-based ionic liquids with high heavy metal extraction rates and further synthetic modifications as well as stripping experiments towards their potential applications are ongoing.Supplementary material 1 (DOCX 222 kb)Below is the link to the electronic supplementary material.


A receptors is involved in cognitive and motor impairment in HE and that reducing this activation restores these functions. We have developed a new compound, GR3027, that selectively antagonizes the enhanced activation of GABAA receptors by neurosteroids such as allopregnanolone and 3α,21-dihydroxy-5α-pregnan-20-one (THDOC). This work aimed to assess whether GR3027 improves motor incoordination, spatial learning, and circadian rhythms of activity in rats with HE. GR3027 was administered subcutaneously to two main models of HE: rats with chronic hyperammonemia due to ammonia feeding and rats with portacaval shunts (PCS). Motor coordination was assessed in beam walking and spatial learning and memory in the Morris water maze and the radial maze. Circadian rhythms of ambulatory and vertical activity were also assessed. In both hyperammonemic and PCS rats, GR3027 restores motor coordination, spatial memory in the Morris water maze, and spatial learning in the radial maze. GR3027 also partially restores circadian rhythms of ambulatory and vertical activity in PCS rats. GR3027 is a novel approach to treatment of HE that would normalize neurological functions altered because of enhanced GABAergic tone, affording more complete normalization of cognitive and motor function than current treatments for HE.Hepatic encephalopathy (HE) is one of the primary complications of liver cirrhosis. Current treatments for HE, mainly directed to reduction of ammonia levels, are not effective enough because they cannot completely eliminate hyperammonemia and inflammation, which induce the neurological alterations. Studies in animal models show that overactivation of GABA Increased GABAergic tone induces motor incoordination, and extracellular GABA in cerebellum correlates with motor incoordination in rats  impairs spatial learning and memory in the Morris water maze   . Allopre, THDOC) , and all, THDOC)  or porta, THDOC) , 5. Henc, THDOC) , 6. Redumic rats , 17. Howmic rats , 37, 40 mic rats . Finastemic rats .A receptors by neurosteroids such as allopregnanolone and THDOC. The aims were to assess whether GR3027 improves motor incoordination and spatial learning in rats with chronic hyperammonemia or with portacaval anastomosis, two main models of HE recommended by the International Society for Hepatic Encephalopathy -induced HE . Howeveralopathy .A receptors (GR3027 is a 3β-hydroxysteroid within a series of molecules developed and patented by some of the authors (T. Bäckström and G. Ragagnin) for the treatment of CNS disorders (patent WO 2008/063128). 3β-Hydroxysteroids are functional antagonists against 3α-hydroxysteroids that positively modulate GABAeceptors , 42, 43.A receptor subunits, respectively. Cells were detached by trypsin, incubated for 15 min at 37°C in extracellular (EC) solution , and added to the EC solution in the chip bath .HEK293 cells were permanently transfected to constitutively express the human α5, β3, and γ2L or α1, β2, and γ2L GABA2, 10 HEPES, 1.0 EGTA, 2 MgATP, pH 7.2).Whole cell recordings were performed under voltage-clamp conditions. Recordings were performed at room temperature and −17 mV  with an AxonPatch 200B amplifier and a DigiData 1322A converter. Data were acquired with pCLAMP 9.0 and analyzed with Clampfit 9.0 . Patch electrodes (1.5–4 MΩ) were filled with intracellular solution  and diluted in EC solution; at measurements all solutions contained 0.1% DMSO.A receptor subunit combinations studied are present in different parts of neurons . As α1βThe binding of GR3027 was determined for receptors, ion channels, and enzymes, including all major classes of neurotransmitter receptors. A total of 114 targets  were tesMale Wistar rats 140–160 g) were made hyperammonemic by feeding with a diet containing 25% ammonium acetate 0 g were . ControlMale Wistar rats (220–240 g) were subjected to portacaval anastomosis. Rats were anesthetized, and an end-to-side portacaval anastomosis was constructed under aseptic conditions with a continuous suture technique according to the technique of Lee and Fisher . The infAdequate measures were taken to minimize pain and discomfort to the animals. The experiments were approved by the Comite de Experimentación y Bienestar Animal of our Center and performed in accordance with guidelines of the Directive of the European Commission (2010/63/EU) for care and management of experimental animals.1) control rats injected with vehicle, 2) hyperammonemic rats injected with vehicle, 3) control rats injected with 20 mg/kg GR3027, and 4) hyperammonemic rats injected with 20 mg/kg GR3027. Control rats injected with GR3027 were not included subsequently because no relevant effect was found in these rats. In the second set of experiments five groups of rats were used: 1) control rats injected with vehicle, 2) hyperammonemic rats injected with vehicle, and hyperammonemic rats injected with 3) 3, 4) 10, or 5) 20 mg/kg GR3027. Numbers of rats are indicated in figures.GR3027 in sesame oil was administered by daily subcutaneous injections in the back. Two different sets of experiments were performed in hyperammonemic rats. In the first set four groups of rats were used: 1) sham-operated rats injected with vehicle, 2) PCS rats injected with vehicle, and PCS rats injected with 3) 0.7 or 4) 2.5 mg/kg GR3027. Numbers of rats are indicated in figures. The reason that the doses used in the studies with hyperammonemic and PCS rats are different is that GR3027 is metabolized by the liver in rats and we expected a reduced metabolism of GR3027 in PCS compared with control or hyperammonemic rats. For this reason we performed prestudies with different doses of GR3027 in PCS rats to find the doses giving equal exposures in the two rat models. The doses were then reduced in the experiments with PCS rats compared with those with hyperammonemic rats. As shown in In rats GR3027 is metabolized in the liver, and GR3027 dosage adjustments were made in the portacaval shunt (PCS) rats to compensate for the metabolic dysfunction caused by the portacaval shunting. For this a prestudy with different doses was performed, and thereafter the following groups were used: The experimental design, including the treatment period and the time at which the behavioral tests were conducted in the two experimental models, is shown in Motor coordination was tested as described in Gonzalez-Usano et al.  with a wThe test was performed as in Monfort et al.  with a cTraining was composed of three trials per day over 6 days. The task involved locating four pellets placed at the end of different arms according to a random configuration as in Hernandez-Rabaza et al. . The numMotor activity was recorded continuously at intervals of 5 min for 14 days in a 12:12-h light-dark cycle with an actimeter of infrared motion detection as in Ahabrach et al.  using 20 μl of blood.ub) in brain homogenates was determined by dialysis: Fub in hyperammonemia = 0.70% and Fub in PCS = 1.43% (Admescope).At the end of treatment plasma was collected from the tail vein. After death by decapitation brains were collected and frozen on dry ice. For analysis of GR3027, brain tissue was homogenized with a 1-to-4 ratio of tissue to PBS (pH 7.4) and extracted with 2 volumes of methanol-acetonitrile (1:1). Plasma protein was precipitated with 3 volumes of acetonitrile. Analyses were performed with a Waters ACQUITY UPLC+Waters XEVO-TQS triple quadrupole mass spectrometer . For calculations of the amount of free GR3027 in the brain the fraction unbound  were used to evaluate effects of GR3027. SPSS statistical package versions PASW 19.0 and 22 were used for all statistical tests.Each data point has its own control; t-test when only one parameter was compared with GraphPad Prism .Statistical significance was estimated with two-way ANOVA and Bonferroni post hoc test and with Student's A receptor. GR3027 (1 μM) antagonizes the effect of THDOC at both the α1β2γ2L- and α5β3γ2L-subunit variants of the GABAA receptor (A and C). With the α1β2γ2L receptor 1 μM GR3027 inhibits 29 ± 5% of THDOC enhancement of the GABA response (P < 0.001), and with the α5β3γ2L receptor the inhibition is 49 ± 5% (P < 0.001) in the experimental conditions used.The effects of GR3027 were studied with patch-clamp measurements on recombinant HEK293 cells expressing human variants of the GABAreceptor , A and CA receptor (B and E). There is no significant effect of GR3027 at either the α1β2γ2L GABAA receptor  or the α5β3γ2L GABAA receptor  when GABA is the sole activator. GR3027 dose-dependently antagonized THDOC . During the specific conditions used for the different receptors, IC50 = 250 nM for the α1β2γ2L GABAA receptor and 350 nM for the α5β3γ2L GABAA receptor.In contrast, GR3027 (1 μM) does not antagonize the effect of GABA at the GABAreceptor , B and EAt 10 μM GR3027 did not show binding activity at any of the studied neurotransmitter-related receptors, steroid receptors, or peptide receptors in P < 0.05) slips (1.4 ± 0.1) than control rats (1.0 ± 0.1). GR3027 restores motor coordination (P < 0.05) and 20 mg/kg . At 10 mg/kg GR3027 also normalized motor coordination (1.0 ± 0.2 slips), but not significantly  slips (1.2 ± 0.1) than control rats (0.71 ± 0.07). GR3027 also restores motor coordination in PCS rats (P < 0.05 vs. PCS) for 0.7 mg/kg and to 0.8 ± 0.1 (P = 0.058) for 2.5 mg/kg  in hyperammonemic rats, such that in the memory test they remained for less time in the right quadrant (30 ± 2% of time) than control rats (39 ± 2%). GR3027 restored spatial memory in the Morris water maze. The percentages of time spent in the correct quadrant were 41 ± 4%, 42 ± 5%, and 38 ± 3% for 3, 10, and 20 mg/kg doses, respectively  in PCS rats. In the memory test PCS rats remained for less time in the right quadrant (31 ± 3% of time) than control rats (41 ± 2%). GR3027 restored spatial memory. The percentages of time spent in the correct quadrant were 34 ± 4% and 39 ± 3% for 0.7 and 2.5 mg/kg doses, respectively  in hyperammonemic rats (18 ± 3 errors) than in control rats  in PCS rats (22 ± 2 errors) than in sham-operated rats (10 ± 2 errors). Treatment of PCS rats with 0.7 mg/kg GR3027 was not enough to improve performance in the radial maze (23 ± 2 errors). Treatment with 2.5 mg/kg GR3027 completely normalized performance in the radial maze . In PCS rats, therefore, treatment with 0.7 mg/kg GR3027 is not enough to restore spatial learning, while 2.5 mg/kg restores it  than in control rats . GR3027 at 0.7 mg/kg increased (P < 0.05) the activity in PCS rats to 2,652 ± 275 counts. At 2.5 mg/kg GR3027 did not affect ambulatory counts  (PCS rats show reduced motor activity (ambulatory counts) during the night (active phase for rats), showing 1,849 ± 176 counts, which is lower ( counts) B.P < 0.001) in PCS rats to 0.8 ± 0.16. GR3027 improved (P < 0.05) the night-to-day ratio of activity, reaching 1.7 ± 0.2 and 1.6 ± 0.3 for 0.7 and 2.5 mg/kg, respectively. This indicates partial restoration of circadian rhythm of activity.The night-to-day ratio of ambulatory activity was reduced in PCS rats, indicating altered circadian rhythm D. This rA and C). GR3027 at 20 mg/kg showed a tendency to slightly increase the activity and the ratio, but the effect did not reach statistical significance.Hyperammonemic rats also showed reduced ambulatory activity during the night and night-to-day ratio of activity compared with control rats , A and CP < 0.05) than in control rats . GR3027 at 0.7 mg/kg and 2.5 mg/kg did not affect nocturnal vertical activity  , showing 561 ± 108 counts, which is lower (ctively) F.P < 0.05) than in control rats (391 ± 64 counts). GR3027 at 0.7 mg/kg and 2.5 mg/kg completely normalized vertical activity during the day, reaching 339 ± 47 and 424 ± 44 counts, respectively . GR3027 improved (P < 0.01) the night-to-day ratio of activity, reaching 2.1 ± 0.4 and 1.9 ± 0.6 for 0.7 and 2.5 mg/kg, respectively . GR3027 at 20 mg/kg did not affect the activity or the ratio.Hyperammonemic rats also showed reduced vertical activity during the night and night-to-day ratio of activity compared with control rats , E and GP < 0.001) in hyperammonemic rats (167 ± 16 μM) compared with control rats (47 ± 3 μM). GR3027 did not affect ammonia levels in hyperammonemic rats (139 ± 14 μM).Blood ammonia levels were increased (P < 0.001) in PCS rats (411 ± 39 μM) compared with sham-operated rats (51 ± 13 μM). GR3027 did not affect blood ammonia, which remained at 380 ± 22 and 348 ± 75 μM in PCS rats treated with 0.7 and 2.5 mg/kg GR3027, respectively.Blood ammonia levels were also increased (In hyperammonemic rats the once-daily administration of GR3027 at 3, 10, and 20 mg/kg resulted in a dose-dependent exposure in plasma and brain. At the time of behavioral testing the total concentrations of GR3027 in plasma were 0.34 ± 0.03, 1.08 ± 0.11, and 1.95 ± 0.61 μM, respectively, and in the brain tissue the unbound concentrations of GR3027 were 6.1 ± 1.4, 11.6 ± 1.4, and 23 ± 5 nmol/kg, respectively .−1·day−1, respectively, and unbound concentrations in the brain were 6.18 ± 0.97 and 17 ± 2 nmol/kg, respectively (In PCS rats the exposures were also dose dependent, and with the lower doses used in these rats, 0.7 and 2.5 mg/kg, the exposures were very similar to those in the hyperammonemic rats. At the time of behavioral testing total concentrations in plasma were 0.48 ± 0.09 and 1.64 ± 0.30 μM, at 0.7 and 2.5 mg·kgectively .These data clearly show that alteration of liver function or portosystemic shunts affects the systemic dose of the drug to be used to reach the levels obtained in the absence of liver failure. The data in A receptor restores motor coordination and spatial learning and memory in rats with HE. GR3027 also partially restores the circadian rhythm of motor activity. Similar beneficial effects of GR3027 would be expected in the motor coordination and cognitive function of patients with HE.We show for the first time that a selective inhibitor of positive neurosteroid modulation of the GABAA receptors.Restoration of motor coordination by GR3027 would be due to a reduction of the GABAergic tone in cerebellum. Increased GABAergic tone in cerebellum induces motor incoordination, and the extracellular GABA concentration in cerebellum correlates with motor incoordination in rats . In hypeA receptors. Overactivation of GABAA receptors in the hippocampus impairs spatial learning and memory in different situations (We show that GR3027 restores spatial learning and memory in the radial and Morris water mazes in hyperammonemic and PCS rats. Impairment of learning and memory would be due to enhanced GABAergic tone in the hippocampus, and restoration by GR3027 would be due to reduction of the neurosteroid-induced activation of GABAtuations , 39, 41.tuations , 31.A receptors during inflammation (A receptors are involved in benzodiazepine-induced decreased learning and memory in the Morris water maze (A receptors might be involved in restoration of learning and memory in HE. This agrees with our previous report showing that impairment of spatial learning in the Morris water maze by acute GABAA receptor activation by neurosteroid treatment is improved by the neurosteroid antagonist UC1011 (Enhanced activation of α5-containing GABAammation  or by etammation  impairs ammation . Both α1ter maze . Thus GRt UC1011 .A receptor activity. As the levels of these neurosteroids and the expression of GABAA receptor subtypes are different in different brain areas (The results reported show that in PCS rats treatment with 0.7 mg/kg GR3027 is enough to restore motor coordination B but notin areas , 45, difGR3027 also partially improves the alterations in circadian rhythms of activity in rats with HE. The mechanisms underlying these alterations are not well known, and it would be speculative to discuss the mechanisms underlying the beneficial effects of GR3027. Nevertheless, altered circadian rhythms of activity are associated with altered sleep in PCS rats , which rA receptor-modulating steroid antagonist, GAMSA, that is, a compound that antagonizes the neurosteroid enhancement of GABAA receptor activation but is without effect when activating steroids as allopregnanolone and THDOC are not present. Thus GR3027 does not antagonize the effect of GABA, which is preferable from a safety perspective as there will be no risk for seizure induction by GR3027. This is different from other GABAA receptor active substances with beneficial effects on cognition in HE models (A receptor but effectively antagonizes benzodiazepine effects (GR3027 is a GABAE models , 17, as E models . FlumazeA receptor activation. Therefore GR3027 may normalize GABAergic neurotransmission and restore neurological functions altered because of overactivation of GABAA receptors, even if hyperammonemia and inflammation associated with chronic liver disease remain present. Thus GR3027 may afford a more complete normalization of motor coordination and cognitive function by acting on a step that mediates the effects of hyperammonemia and inflammation on neurological functions.The main advantage of GR3027 over currently used drugs for treating HE in patients is that GR3027 acts on a brain target, by modulating GABAUmecrine Cognition AB.This study was financed by This study was financed by Umecrine Cognition AB. All authors therefore have this conflict of interest.Author contributions: M.J., M.D., T.B., and V.F. conception and design of research; M.J., M.D., and V.F. interpreted results of experiments; M.J., M.D., T.B., and V.F. edited and revised manuscript; A.A., M.L., C.M., J.S., E.M., and G.R. performed experiments; A.A., M.L., C.M., J.S., E.M., G.R., and V.F. analyzed data; A.A. and M.L. prepared figures; M.D., T.B., and V.F. approved final version of manuscript; V.F. drafted manuscript.


The clinical utility of primaquine (PQ), used as a racemic mixture of two enantiomers, is limited due to metabolism-linked hemolytic toxicity in individuals with genetic deficiency in glucose-6-phosphate dehydrogenase. The current study investigated differential metabolism of PQ enantiomers in light of the suggestions that toxicity and efficacy might be largely enantioselective.13C-labelled primaquine and its two enantiomers (+)-PQ, (−)-PQ were separately incubated with cryopreserved human hepatocytes. Time-tracked substrate depletion and metabolite production were monitored via UHPLC–MS/MS.Stable isotope −1; intrinsic clearance (Clint) of 2.55 and 8.49 (µL/min)/million cells, which when up-scaled yielded Clint of 6.49 and 21.6 (mL/min)/kg body mass was obtained respectively for (+)- and (−)-PQ. The extrapolation of in vitro intrinsic clearance to in vivo human hepatic blood clearance, performed using the well-stirred liver model, showed that the rate of hepatic clearance of (+)-PQ was only 45 % that of (−)-PQ. Two major primary routes of metabolism were observed—oxidative deamination of the terminal amine and hydroxylations on the quinoline moiety of PQ. The major deaminated metabolite, carboxyprimaquine (CPQ) was preferentially generated from the (−)-PQ. Other deaminated metabolites including PQ terminal alcohol (m/z 261), a cyclized side chain derivative from the aldehyde (m/z 241), cyclized carboxylic acid derivative (m/z 257), a quinone-imine product of hydroxylated CPQ (m/z 289), CPQ glucuronide (m/z 451) and the glucuronide of PQ alcohol (m/z 437) were all preferentially generated from the (−)-PQ. The major quinoline oxidation product (m/z 274) was preferentially generated from (+)-PQ. In addition to the products of the two metabolic pathways, two other major metabolites were observed: a prominent glycosylated conjugate of PQ on the terminal amine (m/z 422), peaking by 30 min and preferentially generated by (+)-PQ; and the carbamoyl glucuronide of PQ (m/z 480) exclusively generated from (+)-PQ.The initial half-life of 217 and 65 min; elimination rate constants (λ) of 0.19 and 0.64 hMetabolism of PQ showed enantioselectivity. These findings may provide important information in establishing clinical differences in PQ enantiomers. Plasmodium vivax, in addition to its use in the prophylaxis of all forms of human malaria +m/z 577.2423 + 577.2397).d-2,3,4-O-triacetylglucuronide methyl ester (350 mg) was dissolved in a mixture of MeOH/H2O (5:1)(10 mL) stirred at 0 °C, added ethyl di-isopropyl amine (2 mL) and continued stirring overnight at room temperature. Solvent was evaporated under vacuum and the residue was chromatographed on reverse phase silica (C-18) column with H2O:MeOH to give primaquine alcohol-O-β-d-glucuronic acid.Primaquine alcohol-O-β-1H NMR δ (CD3OD): 8.67 , 8.44, 7.64 , 6.48 , 5.14 , 4.52 , 3.88 , 3.84 , 3.68 , 3.40 , 3.34 , 3.20 , 1.80 , 1.30 ; HRESIMS [M + H]+m/z 437.1884 + 437.1924).N-Methyl mopholine (0.25 mL) was added to a mixture of benzyl d-glucuronate , levulinic acid  and HATU  in anhydrous acetonitrile (10 mL) and the reaction mixture was stirred 2 h at ambient temperature. Reaction was quenched with Ambalite H+ resin, filtered and the residue was thoroughly washed with CH2Cl2. Combined organic extract was evaporated to dryness and chromtographed on silica column with CH2Cl2:MeOH (96:4) to give benzyl 1-O-levulinyl-d-glucuronate (550 mg).1H NMR δ (CDCl3): 2.06 , 2.72-2.48 , 3.80-3.58 , 4.02 , 5.15 , 5.60 , 7.30 .4 was added portion wise to a mixture of benzyl 1-O-levulinyl-d-glucuronate  and 6-methoxy-8-aminoquinoline  in glacial acetic acid (5 ml) at 20 °C under stirring while monitoring the reaction by TLC. Upon completion, the reaction was quenched with ice/water, neutralized with 10 % NaOH and extracted with CH2Cl2. The organic extract was dried on anhydrous Na2SO4, evaporated to dryness. The product was purified by chromatography on silica gel and elution with CH2Cl2/MeOH (5 %) to give carboxyprimaquine-β-d-glucuronide benzyl ester (260 mg).NaBHd-glucuronide benzyl ester (210 mg) and 10 % Pd/C in EtOH (10 mL) was hydrogenated with H2 (20 psi) for 2 h. Reaction mixture was filtered through Celite purified by silica column chromatography with CH2Cl2:MeOH to give carboxyprimaquine-β-d-glucuronide (140 mg).Carboxyprimaquine-β-1H NMR δ (CD3OD): 8.48 , 8.02 , 7.35 , 6.38 , 5.51 , 3.91 , 3.84 , 3.68 , 3.46 , 3.39 , 3.30 , 2.54 , 1.90 , 1.25 : 13C NMR δ (CD3OD): 19.1, 30.2, 30.8, 54.3, 71.6, 72.1, 75.5, 76.0, 94.0, 97.4, 121.5, 130.1, 135.1, 143.8, 144.5, 159.5, 172.4: HRESIMS [M + H]+m/z 451.1776 + 451.1717).3CN (5 mL) was added dropwise to a solution of di-tert-butyl dicarbonate  in anhydrous CH3CN (5 mL) at 0 °C under stirring. After 10 min, a solution of (+)-(S)-primaquine free amine  in anhydrous CH3CN (5 mL) was added dropwise and the reaction mixture was stirred for further 30 min at 0 °C. Solvent was evaporated under reduced pressure and the resulting dark yellow oily residue was dissolved in toluene (5 mL), cooled to 0 °C, treated dropwise with a solution of methyl 2,3,4-tri-O-acetyl-d-glucuronnte  in toluene (5 mL) followed by triethylamine (1.2 mL) and stirred for 2 h at 0 °C and another 2 h at room temperature. Solvent was evaporated under reduced pressure and the dark brown residue was chromatographed on silica column and eluted with ethyl acetate/hexane (40:60) to give (S)-primaquine-N-carbamoyl -2,3,4-O-triacetyl-β-d-glucuronide methyl ester (1.80 g).A solution of 4-dimethylamino pyridine  in anhydrous CH1H NMR δ (CDCl3): 1.25 , 1.60 , 1.99 , 3.15 , 3.58 , 3.66 , 3.85, 4.12 , 5.31-5.09 , 5.67 , 5.94 , 6.23 , 6.30 , 7.27 , 7.88 , 8.49 ; HRESIMS [M + H]+m/z 620.2451 + 620.2455).N-carbamoyl-2,3,4-O-triacetyl-β-d-glucuronide methyl ester  in MeOH (30 mL) and H2O (6 mL) at 0 °C while stirring. After 10 min at 0 °C, cooling bath was removed and stirring was continued overnight. Solvent was removed under reduced pressure and the residue was dissolved in MeOH (2 mL), introduced to a Sephadex column (4 × 30 cm) and eluted with methanol (0.5 mL/min) to give (S)-primaquine-N-carbamoyl-β-d-glucuronide (284 mg) as a pale yellow powder.Ethyldiisopropyl amine (6 mL) was added to a solution of primaquine-1H NMR δ (D6-DMSO): 1.18 , 1.60 , 3.33-2.99 , 3.66 , 3.59 , 3.80 , 5.16 , 6.11 , 6.24 , 6.44 , 7.40 , 8.05 , 8.51 ; 13C NMR δ (D6-DMSO): 20.6, 26.6, 33.8, 47.5, 55.4, 72.3, 72.8, 74.8, 77.1, 92.1, 95.4, 96.6, 122.6, 130.0, 135.0, 135.3, 145.1, 144.7, 155.6, 159.4, 173.1; HRESIMS [M + H]+m/z 480.1954 + 480.1982).Pooled mixed-gender cryopreserved metabolism-qualified  primary human hepatocytes  sourced from free donors  were purchased from Corning Life Sciences  and stored in liquid nitrogen until use.g, 5 min), the recovery medium discarded and the cells were re-suspended in the plating medium. Viability of the suspended hepatocytes was computationally determined based on cell counts using a Bio-Rad automated cell counter . The cell density was adjusted to approximately 1X106 viable cells/mL in the plating media.The cryopreserved hepatocytes were thawed and the cells were suspended in recovery medium (Corning Life Sciences). According to the supplier’s instructions, the suspended cells were centrifuged  attached with a shaker set at 75 rpm. A separate control incubation of PQ and the two enantiomers in the cell-free hepatocyte media was performed to detect any nong, 10 min) and clear supernatants were dried using a Speedvac. The dried samples were re-suspended in 150 µL methanol, centrifuged and clear supernatants were transferred to HPLC sample vials for analysis.In preparation for HPLC–MS analysis, samples were centrifuged  on an ACQUITY UPLC system  to which a conditioned auto-sampler (at 20 °C) was attached. Details of the chromatography set-up are published , the hepatic metabolic blood clearance  was predicted using the well-stirred model: CLh,b = (CLint in vivo × Q)/(CLint in vivo + Q) [Initial half-life (tncubates : CLint iivo + Q) .Since this estimation was for comparisons between enantiomers only, no corrections were made for the free fractions in vitro or in vivo—i.e. they were assumed to be identical .Intra- and inter-day variations in the data obtained from replicated experiments were assessed for significance. Replicate agreements were within 5 % variations. All results were statistically analyzed for significance by Student’s t test using GraphPad Prism .PQ was incubated in vitro with human hepatocytes (1 million cells/mL) at a final concentration of 20 µM. Substrate concentrations, as initially titrated, showed optimal metabolism at 20 µM (Km = 37 µM). Although this is higher than expected peak plasma concentrations in humans as we earlier reported (<2 µM) , it apprSubstrate depletion and metabolite production were monitored via UHPLC-MS/MS. By 2 h, 30, 16 and 42 % of PQ, (+)-PQ and (−)-PQ, respectively, were depleted /million cells, which when upscaled in vivo based on known human hepatocellularity (hepatocytes/g liver) and liver mass (g/kg body mass) yielded 6.49 and 21.6 (mL/min)/kg body mass respectively for (+)- and (−) PQ. Extrapolation to in vivo human hepatic blood clearance was performed using the well-stirred liver model, which showed that the rate of hepatic clearance of (+)-PQ was only 30 % that of (−) PQ - and (−)-PQ, with elimination rate constants (ʎ) of 0.19 and 0.64 hFrom the metabolite profile, two major routes of metabolism were observed as earlier reported: oxidative deamination of the side chain terminal amine; and hydroxylations on the quinoline moiety . The metN-aminoalkyl side chain of PQ to an intermediate PQ aldehyde which is further converted to the carboxylic acid by an aldehyde dehydrogenase. The PQ aldehyde formed from PQ may also undergo a reduction reaction to form the primaquine alcohol -PQ.A major product of the quinoline ring oxidation identified was the one that corresponds to an PQ Figs. d. Site om/z 422; Fig. -enzymatically in the cell-free media -PQ -PQ (about 8 times higher than in (+)-PQ at the early time points). These results support our previous data on in vivo pharmacokinetics of PQ enantiomers in rodents, primates and humans as well in vitro results with freshly isolated human hepatocytes [N-aminoalkyl side chain were consistently more abundant from the (−)-PQ than (+)-PQ. A recent pharmacokinetic study in human [The results presented herein have predicted that the initial half-life (as well as other pharmacokinetic parameters derived therefrom) of (+)-PQ was more than three times longer than that that of (−)-PQ. MAO and CYP are known to be involved in the phase I hepatic metabolism of PQ . A majoratocytes , 15. It m/z 274 and 289 respectively). Traces of other hydroxylated metabolites were observed, including the 2-OH and 4-OH-PQ, and three other quinone imines (m/z 274). The position of the carbonyl group of these metabolites could not be determined by mass analysis. Stability of the keto-imines however, suggests that the keto group is unlikely to be present at C-5 of the quinoline ring, as for this metabolite, a rapid degradation with demethylation of the 6-methoxy is observed.Only two ring-hydroxylated PQ metabolites attributable to CYP pathway were present in substantial amounts. They were identified by accurate mass as quinone-imine metabolites of hydroxylated PQ and CPQ - and (−)-PQ, respectively) were observed during a 2 h incubation period, and CYP2D6 preferentially converted (+)-PQ to its PQ-5,6-orthoquinone and 2-hydroxyprimaquine . MeanwhiN-carbamoyl glucuronide of PQ accounted for most of the (+)-PQ consumption. Identity of N-carbamoyl glucuronide of PQ was confirmed by comparison of its HPLC retention time and MS/MS fragmentation properties with the synthetic standard. It accumulated linearly over time and was the major product after prolonged incubation of PQ with human hepatocytes. The formation of this carbamoyl glucuronide of PQ as a putative metabolite in the incubation mixture of racemic PQ with pooled human hepatocytes was previously reported [N-carbamoyl glucuronides are unusual, but have been reported as minor products for other drugs containing primary and secondary amines [2 environment with UDP-glucuronyltransferases can readily yield the carbamoyl glucuronides. It is, however, somewhat surprising that in the hepatocytes, this product is enantioselectively formed.The formation of the phase II conjugation reaction metabolite, identified as an reported . In the y amines , 28. IncAnother unexpected metabolite observed was an apparent glucose conjugate of PQ. The biphasic kinetics of formation of PQ-glucose conjugate suggests that formation of this metabolite may also depend on other metabolic reactions in the hepatocytes. It would be interesting to further investigate the pathway(s) involved in formation of this metabolite. The earlier small peak of formation of PQ-glucose conjugate may not be enzyme-dependent, since formation of this conjugate could be observed even in cell-free control incubations.As observed against time of incubation, metabolite profiles showed different shapes, suggesting variation in kinetics and disposition. The interplay of factors including multiple enzyme involvement, dependence of one metabolite on the formation of the other, possible interactions among the metabolites might be responsible for the varying kinetics of formation of the different metabolites.N-8 alkyl amino side-chain. Overall, the ring oxidation products appeared to be formed in limited amounts. A phase II conjugation reaction metabolite N-carbamoyl glucuronide of PQ was identified as a major metabolite, which was exclusively formed from (+)-PQ. Another PQ conjugate formed by apparent N-glucosylation was preferentially formed with (+)-PQ. These findings enhance the understanding of the pathways of PQ metabolism, and further inform the enantioselectivity of its metabolic profiles.The enantiomers of PQ showed significant variation in in vitro pharmacokinetics as well as metabolite generation in human hepatocytes. The in vitro clearance of (−)-PQ is three times faster than that of (+)-PQ in hepatocytes. Trace amounts of ring oxidation products were observed with (+)-PQ while (−)-PQ was preferentially metabolized by oxidative deamination of the terminal amine on the


The results revealed that the polyacrylic acid content had great influence on the morphology of 0.7PMN-0.3PT. With increasing PAA concentration from 0.45 to 0.82 g/ml, the ratio of perovskite phase (PMN-PT nanorod) increased, while the ratio of pyrochlore phase decreased. When the PAA concentration was 0.82 g/ml, pure 0.7PMN-0.3PT nanorods were obtained. However, when PAA concentration was higher than 0.82 g/ml, the excess of PAA would hindered their [100] orientation growth. The piezoelectric coefficient d33 of 0.7PMN-0.3PT nanorod was obtained by linear fitting, and the d33 value was 409 pm/V.The effects of surfactant polyacrylate acid (PAA) on shape evolution of 0.7Pb(Mg Then, a drop of suspension was put on Au/Ti-covered silicon wafer and dried in air. The 81 pm/V) . It is wSingle crystalline PMN-PT nanorods of cuboid morphology were obtained at 235 °C with the addition of PAA in hydrothermal reaction. As PAA concentration increased from 0.45 to 0.82 g/ml, the length of the PMN-PT nanorod increased while the growth in the radial direction was hindered. An almost pure perovskite phase (PMN-PT nanorod) appeared at a PAA concentration of 0.82 g/ml. The orientation growth of PMN-PT was attributed to the surface energy difference caused by absorption of PAA on different crystal plane. The piezoelectric coefficient of 0.7PMN-0.3PT nanorod was 409 pm/V, which provides a great potential application in manufacturing piezoelectric nanodevices.


Neosartorya fischeri P1, termed NfBGL1, capable of efficiently converting isoflavone glycosides into free isoflavones. The gene, belonging to glycoside hydrolase family 3, was successfully overexpressed in Pichia pastoris at high cell density in a 3.7-l fermentor. Purified recombinant NfBGL1 had higher specific activity (2189±1.7 U/mg) and temperature optimum (80°C) than other fungal counterparts when using p-nitrophenyl β-d-glucopyranoside as the substrate. It retained stable at temperatures up to 70°C and over a broad pH range of 3.0−10.0. NfBGL1 had broad substrate specificity including glucosidase, cellobiase, xylanase and glucanase activities, and displayed preference for hydrolysis of β-1,2 glycosidic bond rather than β-1,3, β-1,4, β-1,6 bonds. The enzyme showed high bioconversion ability for major soybean isoflavone glycosides  into free forms. These properties make NfBGL1 potential for the wide use in the food, feed, pharmacy and biofuel industries.Isoflavone occurs abundantly in leguminous seeds in the form of glycoside and aglycone. However, isoflavone glycoside has anti-nutritional effect and only the free type is beneficial to human health. In the present study we identified a β-glucosidase from thermophilic  Plant isoflavones are important secondary metabolites that are mainly distributed in leguminous plants with high levels http://www.cazy.org) Dalbergia sp. Paecilomyces thermophilaDictyoglomus turgidumSulfolobus solfataricusRhizopus spp. β-Glucosidases (EC3.2.1.21) catalyzes the hydrolysis of β-glucosidic linkages of various oligosaccharides and glycosides to release glucose and debranched oligosaccharides. According to the CAZy database  and other p-nitrophenyl glycosides, and Novozyme 188 (cellobiase from Aspergillus niger) were purchased from Sigma-Aldrich . Other substrates including barley β-glucan and xyloglucan were obtained from Megazyme . T4 DNA ligase and restriction endonucleases were purchased from Promega .The substrates cellobiose, carboxymethyl cellulose sodium (CMC-Na), Avicel, laminiarin, lichenin, amygdalin, daidzin, genistin, glycitin, daidzein, genistein, glycitein, birchwood xylan, beechwood xylan, sophorose, gentiobiose, salicin, lactose, The Fungal DNA Isolation Kit and RNeasy Plant Mini Kit were obtained from Omega Bio-tek  and QIAGEN , respectively. The DNA Purification Kit, LA Taq DNA polymerase and restriction endonucleases were purchased from TaKaRa . All other chemicals were of analytical grade and commercially available.N. fischeri P1 CGMCC 3.15369 4)2SO4, 1 g/l KH2PO4, 0.5 g/l MgSO4·7H2O, 0.2 g/l CaCl2, and 10 mg/l FeSO4·7H2O at 45°C and pH 5.0 for 4 days. The total β-glucosidase activity in N. fischeri culture supernatants was 11.9±0.2 U/ml.Escherichia coli Trans1-T1  was cultivated in Luria-Bertani (LB) medium with 100 µg/ml ampicillin at 37°C for gene cloning and sequencing. P. pastoris GS115  cultivated in yeast peptone dextrose (YPD) medium at 30°C was used for heterologous protein expression. The plasmids pEASY-T3 (TransGen) and pPIC9 (Invitrogen) were used as cloning and expression vectors, respectively.invitro using the ReverTra Ace-α- Kit  with the total RNA as the template. According to the genome sequence of a hypothetical β-glucosidase from N. fischeri NRRL 181 (XP_001261562), two specific primer sets  were designed to amplify the full-length β-glucosidase gene, Nfbgl1, and its cDNA without the signal peptide coding sequence. The PCR products with appropriate size were ligated into a pGEM-T3 Easy vector (TransGen) for sequencing.Mycelia of strain P1 were collected after 4 days’ growth in β-glucosidase producing medium as described above. Total DNA and RNA were extracted and purified according to the manufacturer’s instructions, respectively. The cDNAs were synthesized http://www.ncbi.nlm.nih.gov/BLAST/) and AlignX from Vector NTI were used to analyze the nucleotide and deduced amino acid sequences, respectively. The online software FGENESH (http://linux1.softberry.com/berry.phtml) was used to predict the transcription initiation sites, introns and exons. NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) was used to predict the potential N-glycosylation sites. Multiple sequence alignment of deduced NfBGL1 and other fungal counterparts were conducted using the ClustalW program . The three-dimensional structure was predicted using the SWISS-MODEL (http://swissmodel.expasy.org/) with the GH3 β-glucosidase AaBGL1 from Aspergillus aculeatus  The nucleotide sequence was analyzed using Vector NTI Advance 10.0 software (Invitrogen). BLASTn and BLASTp programs (Pichia Expression Kit (Invitrogen). The cDNA fragment of mature NfBGL1 without the signal peptide-coding sequence was digested with EcoRI and NotI, and then cloned into the corresponding sites of vector pPIC9. The recombinant plasmid pPIC9-Nfbgl1 was then linearized with BglII and transformed into P. pastoris GS115 competent cells by electroporation . The positive transformants were selected based on their enzymatic activities in shake tubes. The transformant that exhibited the highest β-glucosidase activity in culture supernatant was used for large-scale fermentation in 1-l Erlenmayer flasks. The cell-free culture supernatants were collected and loaded onto a HiTrap Q Sepharose XL FPLC column  equilibrated with buffer A . Proteins were eluted with NaCl gradients (0–1.0 M) in the same buffer at a flow rate of 3 ml/min. The fractions containing enzyme activity were collected and evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was determined by the Bradford assay with bovine serine albumin as the standard.Heterologous expression of recombinant NfBGL1 was conducted following the The positive transformant with the highest β-glucosidase activity in shake flask culture was selected for high-cell-density fermentation in a 3.7-l stirred-tank fermentor  using a three-step fermentation strategy as described pNPG as the substrate. The reaction system consisted of 400 µl of 1.25 mM pNPG in McIlvaine buffer  and 100 µl of appropriately diluted enzyme solution. The mixture was incubated at 80°C for 10 min, and then terminated by addition of 1.5 ml of 1 M Na2CO3. After cooling down to room temperature, the absorbance was measured at 405 nm. One unit of enzyme activity was defined as the amount of enzyme that released 1 µmol of p-nitrophenol per min. Each reaction was performed in triplicate, and the data were shown as mean ± S.D. (n = 3). The enzymatic activities towards other p-nitrophenyl glycosides were assayed as described above with different substrates.The β-glucosidase activity was assayed using Cellulase/glucanase/xylanase activity was determined according to the 3,5-dinitrosalicylic acid (DNS) assay The enzymatic activities on 10 mM of cellobiose, salicin, lactose, daidzin, glycitin, and genistin were estimated by measuring the amount of released glucose using GOD-POD method with a commercial kit . The absorbance was measured at 505 nm. One unit of enzyme activity was defined as the amount of enzyme that released 1 µmol of glucose per minute.The optimal pH values were determined at 80°C in the following solutions, 100 mM glycine-HCl for pH 2.0–3.0, McIlvaine buffer for pH 3.0–8.0, 100 mM Tris-HCl for pH 8.0–9.0, and 100 mM glycine-NaOH for pH 9.0–12.0. The pH stability was determined by measuring the residual enzyme activity under the standard conditions  after pre-incubation of the enzyme in buffers of pH 2.0–12.0 at 37°C for 1 h without the substrate.The optimal temperature was examined at optimal pH by measuring the enzyme activity over the temperature range of 20 to 90°C. The thermostability assay was carried out by incubating the enzyme at optimal pH and at 70°C or 80°C without substrate for 2, 5, 10, 20, 30, or 60 min, and then measuring the residual enzyme activities under the assay conditions.+, K+, Li+, Ag+, Cu2+, Ni2+, Mn2+, Ca2+, Pb2+, Co2+, Zn2+, Mg2+, Fe3+, Cr3+, SDS, EDTA, and β-mercaptoethanol was subject to enzyme activity assay under the standard conditions and compared to the blank control without any additives.To investigate the effects of different metal ions and chemical reagents on the activity of purified recombinant NfBGL1, the reaction system containing 5 mM of each Nap-nitrophenyl derivatives and oligosaccharides, or 10 mg/ml of polysaccharides and isoflavone glycosides.The substrate specificities of NfBGL1 were determined under standard assay conditions (pH 5.0 and 80°C for 10 min) in the presence of 1 mM of Km and Vmax values of NfBGL1 were determined in McIlvaine buffer (pH 5.0) containing 0.01–2 mM pNPG at 80°C for 5 min. The experiments were carried out three times, and each experiment included three replicates. The data were calculated using the Lineweaver-Burk method with a non-linear regression computer program GraFit .The pNPG and 250 µl of McIlvaine buffer (pH 5.0) containing the same amount of glucose. The residual enzyme activity was measured at 80°C, pH 5.0 for 10 min. The Ki value was calculated by plotting the reciprocal of the reaction velocity and inhibitor concentration at each pNPG concentration.The inhibitory effect of glucose on NfBGL1 activity was determined by fitting to the Dixon plot g, 4°C for 10 min, 20 µl of each supernatant was subject to HPLC analysis by using Waters HP1100 HPLC system  equipped with a C18 column . The solvent was composed of acetonitrile and 100 mM phosphate buffer (pH 5.0) at the ratio of 70∶30 (v/v) and run at a flow rate of 0.8 ml/min. The chromatograms were detected at 260 nm.To compare the hydrolysis efficiencies of NfBGL1 and Novozyme 188 towards isoflavone glycosides, 50 µl of 10% (w/v) soybean flour was incubated with 200 µl of each enzyme solution (0.05 U) in McIlvaine buffer (pH 5.0) in a thermostatically controlled incubator at 50°C. The reactions were terminated by ice water cooling. After centrifugation at 13,000 Nfbgl1 from N. fischeri P1 consists of 2220 base pairs and encodes a polypeptide of 739 amino acids with a putative signal peptide of 17 residues at the N-terminus. The molecular mass and pI value were estimated to be 78.8 kDa and 5.54, respectively. Two putative N-glycosylation sites (Asn207 and Asn381) were identified in deduced NfBGL1.The cDNA of N. fischeri NRRL 181 (XP_001261562), and of 61% identity with the Trichoderma reesei β-glucosidase (1713235A) with the experimentally evidenced activity Hordeum vulgareKluyveromyces marxianusThe deduced amino acid sequence of mature NfBGL1 showed the highest identity of 100% with the hypothetical β-glucosidase from Nfbgl1 without the signal peptide-coding sequence was amplified by PCR and cloned into the pPIC9 vector for heterologous expression. The β-glucosidase activity of recombinant NfBGL1 was determined to be 33.5±0.4 U/ml  after 2-day growth in 1-l Erlenmayer flasks at 30°C. The culture supernatant was purified to electrophoretic homogeneity by one-step anion exchange chromatography. A single band of approximately 80 kDa migrated in SDS-PAGE . Considering its acidic, thermophilic, wide pH/temperature range-stable and chemical-resistant advantages in biomass conversion, handling, post-processing and transportation Generally fungal β-glucosidases have an acidic pH optimum, ranging from 4.0 to 6.5, and are usually stable over a wide pH range . NfBGL1  4.5–5.5 . In comp 4.5–5.5 . NfBGL1 pNPG, it was active towards the β-linked synthetic substrate p-nitrophenyl β-d-cellobioside and p-nitrophenyl β-d-xylopyranoside. Of all tested saccharides, sophorose  was the most preferred substrate, followed by laminiarin , salicin (β-linked), cellobiose , lichenan , and lactose . Although most β-glucosidases have higher activities toward β-1,4-linked saccharides, NfBGL1 as well as the β-glucosidases from Trichoderma koningiiNfBGL1 exhibited broad substrate specificity including β-glucosidase, cellobiase, glucanase, xylanase and isoflavone glycoside hydrolysis activities . BesidespNPG as the substrate, the Km, Vmax and kcat values for NfBGL1 was 0.51 mM, 2172 µmol/min/mg, and 2853/s, respectively. The Km value of NfBGL1 fell within the range of fungal β-glucosidase Km values, but its Vmax value was quite high (kcat/Km) is the true kinetic parameter to evaluate enzyme catalytic performance. The kcat/Km value of NfBGL1 was 5594/mM/s, higher than that of β-glucosidases from P. thermophila (42.8/mM/s) Stachybotrys sp. (494.6/mM/s) C. thermophilum (193.4/mM/s) With ite high . CatalytCandida peltataAspergillus oryzaeKi values of 1.4 and 1.36 M, respectively. Compared with these two fungal counterparts, NfBGL1 showed less resistance to glucose inhibition with a Ki of 13.4 mM, but the glucose tolerance was much higher than the β-glucosidases from T. reesei, F. palustris, and P. funiculosum NCL1  and isoflavone aglycones  . After 5N. fischeri P1. The enzyme had an optimum temperature and specific activity higher than that of all known fungal counterparts, was stable over a broader pH and temperature ranges, and had strong resistance to most chemicals tested. Its substrate specificity was remarkably broad, including glucosidase, cellobiase, xylanase and glucanase activities. Its high efficiency to convert isoflavone glycosides of different soybean products into aglycons was much attractive. These characteristics make it a preeminent enzyme candidate in the food, feed, pharmacy, and biofuel industries.In the present study, a highly-active, thermophilic, soybean isoflavone glycoside-degrading β-glucosidase of GH3 was identified in


Pinus peuce Grisebach, Pinaceae in Kosovo. Plant materials were collected from three populations in the Sharri National Park and from four other populations in the Bjeshkët e Nemuna National Park, in Kosovo. Essential oils were obtained by steam distillation and analyzed by GC-FID (Gas Chromatography-Flame Ionization Detection) and GC-MS (Gas Chromatography-Mass Spectrometry). The results showed that the yield of essential oils (v/w dry weight) varied depending on the origin of population and the plant organs and ranged from 0.7 to 3.3%. In total, 51 compounds were identified. The main compounds were α-pinene , β-phellandrene , and β-pinene . HCA  and PCA  were used to assess geographical variations in essential oil composition. Statistical analysis showed that the analyzed populations are grouped in three main clusters which seem to reflect microclimatic conditions on the chemical composition of the essential oils.The principal aim of this study was to analyze the chemical composition and qualitative and quantitative variability of essential oils obtained from seven naturally grown populations of the Pinus species demonstrate primarily antimicrobial, expectorant activities and promote blood circulation [ Pinus spp. are therapeutically used externally or by inhalation to treat different medical conditions, including respiratory disease, common colds, and rheumatic complaints such as muscle and joint pain [ Pinus essential oils have been reported as well [ Pinus sylvestris and Pinus mugo, are published. Turpentine oil from Pinus pinaster is officinal in European Pharmacopoeia, Ph.Eur. 6.2 [ Pinus peuce, also known as Balkan pine or Macedonian pine, is a Balkan endemic conifer tree growing in mountains of Bulgaria, Serbia, Macedonia, Greece, Kosovo, and Montenegro between ca. 600 and 2200 m.a.s.l. [Essential oils represent an important class of constituents among various plant families. Due to the high content and interesting composition of their essential oils, numerous species of the Pinaceae family have been traditionally used in medicine, cosmetics, and the food industry. The essential oils ofculation –3. Herbaint pain –3. Antio as well –12. Pine as well . In the  as well , monograEur. 6.2 . Pinus pm.a.s.l. . The matm.a.s.l. , 17. Pinus peuce and its biological activity has been addressed in recently published papers [ P. peuce in oil samples isolated from shots and cones [ Pinus peuce [ Pinus peuce populations and between Pinus peuce, Pinus heldreichii, and Picea omorica have been compared [ Pinus species from the Balkan region hasve been estimated by flow cytometry [ Pinus peuce has been studied by Zhelev et al. [ P. peuce from Kosovo had already been investigated .Plant material was air-dried in the shade at room temperature and cut into small pieces (<0.5 cm). Separated needles and twigs (only woody parts) were subjected to essential oil distillation. For distillation, 50 g of dry tissue was placed into 0.5 liter of water in a 1 liter flask and distilled at a rate of 3 mL/min in a Clevenger apparatus for 3 h. The samples were stored in the dark at −18°C in the freezer pending further analysis. The yield of essential oil is expressed as a volume/weight percentage, accounted to the air-dried plant material  equipped with the flame ionization detector (FID). The separation was conducted on a HP-5MS column 30 m × 0.25 mm with 0.25  m/z. The separation was conducted with the same column and temperature program as for the analytical GC.GC/MS analyses were performed using an Agilent 7890A GC system coupled to a 5975C MSD (Agilent Technologies). The ionization energy was 70 eV with a mass range of 40–400The Kovats Retention Indexes were experimentally determined and compared with those from the literature . The cal P. peuce. PCA and HCA analyses were performed using the statistical analysis software, XLSTAT Version 2014.2.03 . The oil components with concentrations higher than 1% of the total oil amount in twigs and/or needles were subjected to statistical analyses  and Principal Component Analyses (PCA) were used to evaluate whether the identified essential oils components can be useful for reflecting the population diversity of Pinus peuce grown in three populations in Sharri National Park and four populations in Bjeshkët e Nemuna National Park are presented in Tables The chemical composition of essential oils obtained from needles and twigs ofExperimental data revealed that the essential oils were mainly composed of monoterpenes. Their concentration in twigs was higher in comparison to the needles. As presented in Tables α-pinene, β-phellandrene, β-pinene, camphene, and bornyl acetate, as well as the sesquiterpene germacrene.In samples obtained from Sharri National Park and Bjeshkët e Nemuna National Park the dominant constituents were the monoterpenes  P. peuce from the Sharri National Park, α-pinene with an average concentration of 31.6%, was dominant, followed by β-pinene (13.8%), β-phellandrene (9.8%), germacrene D (9.2%), camphene (7.7%) and bornyl acetate (4.4%), while in needles originating from the Bjeshkët e Nemuna National Park the order of the compounds was α-pinene (29.0%) followed by germacrene D (16.6%), β-phellandrene (11.4%), β-pinene (10.9%), bornyl acetate (7.4%) and camphene (5.8%) (In the samples obtained from needles ofe (5.8%) .β-phellandrene (34.4%), followed by α-pinene (17.7%), β-pinene (17.4%), germacrene D (6.5%), bornyl acetate (4.3%), and camphene (3.2%). The order of the twigs samples from Bjeshkët e Nemuna National Park was the same as from previous population: β-phellandrene (45.1%), followed by α-pinene (16.5%), β-pinene (11.0%), germacrene D (7.6%), bornyl acetate (2.6%), and camphene (2.6%)  whereas its concentration in the three populations from the National Park “Bjeshkët e Nemuna” was less than 0.2%. Exceptions were the samples from one population (Roshkodol) with percentages reaching 13.5% in needles and 2.1% in twigs.Twigs from Sharri National Park were dominated by e (2.6%) . Althoug P. peuce growing in Pelister (Macedonia), in twigs with needles and twigs without needles, were the monoterpenes α-pinene , camphene, β-pinene, myrcene, limonene-phellandrene and bornyl acetate, and the sesquiterpenes, (E)-caryophyllene, germacrene D , and δ-cadinene [α-pinene (45.5%), germacrene D (11.1%), β-pinene (10.8%), camphene (10.3%), bornyl acetate (5.0%), β-phellandrene (3.4%), β-caryophyllene (3%), and β-myrcene (0.9%). Nikolić et al. [ P. peuce from three populations from Serbia and Montenegro were α-pinene (36.5%) and germacrene D (11.4%), followed by camphene (8.5%), bornyl acetate (6.8%), β-pinene (6.8%), β-caryophyllene (5.2%), β-phellandrene (4.7%), terpinen-4-ol acetate (1.6%), (E)-hex-2-enal, α-muurolene, β-gurjunene, and β-myrcene. Koukos et al. [ P. peuce in Northern Greece has a similar pattern of constituents as those documented in our study. The dominant constituents in twig oil were α-pinene (7.4%), β-pinene (12.5%), β-phellandrene (27.0%), β-caryophyllene (4.5%), and citronellol (12.5%), whereas the needle oil was rich in α-pinene (23.1%), camphene (5.5%), β-pinene (22.0%), β-phellandrene (6.8%), bornyl acetate (9.7%), β-caryophyllene (3.1%), and citronellol (13.4%). The mean oil yield was 2.9% for twigs and 0.6% for needles. For comparison, in our study the yields of essential oils obtained from twigs were notably higher than those obtained from needles. Average yields of essential oils obtained from samples from the Sharri National Park were in needles 0.85–1.1 and in twigs 2–3.3%, whereas yields from the Bjeshket e Nemuna National Park were in needles 0.8–1.5% and in twigs 1.9–2.7% v/w. Higher yields of twigs comparing to needles have been documented in works of other authors [Experimental results from our study, concerning the composition of essential oils, are in accordance with previously published data. According to Karapandzova et al.  the mostcadinene  identifić et al.  also reps et al.  revealed authors . Pinus peuce from Bjeshket e Nemuna National Park and Sharri National Park are presented.In  P. peuce essential oils, Hierarchical Cluster Analysis HCA .In order to assess the chemical composition ofysis HCA  and Prinysis HCA  were per Pinus peuce shows that there are three main groups. Thus, 3-carene, Z-muurola-4(14),5-diene, myrcene, β-phellandrene, and α-cadinene were the principal components that contributed to the clustering of the samples obtained from needles. E-nerolidol, δ-cadinene, α-terpinyl acetate, germacrene D, bornyl acetate, and β-Z-ocimene were the primary components that contributed to the clustering of the samples obtained from needles of Junik, Hajlë, Liqenat, and Peribreg and twigs from a sample originated from Hajlë and a sample from Liqenat location. The samples obtained from needles of the locations Oshlak, Pashallar, Roshkodol, and Peribreg were dominated by α-pinene, β-caryophyllene, camphene, E-pinene hydrate, terpinolene, α-terpineol, E-piperitol, and myrtenal , which are geographically separated from each other at a distance of approximately 100 km. Due to geographic isolation of the mountainous areas and anemophilous pollination of the Pinus peuce Griseb. growing in seven locations in Kosovo has been elucidated. Experimental results revealed that the dominant constituents of oils were monoterpenes, whereas sesquiterpenes and diterpenes were present in lower amounts. α-Pinene, β-phellandrene, β-pinene, germacrene D, and bornyl acetate were dominant constituents of the essential oils. Statistical analysis of PCA and HCA documented that variability exists in the composition of essential oils and that this is primarily related to the plant organ source of the essential oil, rather than interspecies variation between different populations. Variability in chemical composition of essential oils among populations of P. peuce seems to reflect the environmental impact on these compositions, which is influenced by differences in microclimatic conditions. To confirm the natural variability and chemopolymorphism of this species in Kosovo, further investigation is warranted and should be corroborated with more comprehensive molecular analysis.In this study, the chemical variability of essential oils obtained from needles and twigs of


Every day we see patients coming after some kind of brace treatment for adolescent idiopathic scoliosis (AIS). Literature is made mainly by studies about untreated patients, so we don’t know what to expect when we start such a treatment.The aim of the present study was to compare the results of previously treated scoliosis compared to untreated ones.Prospective observational controlled cohort study nested in a prospective database started in March 2003.Inclusion criteria: patients that started a brace treatment at their first clinical evaluation at our institute between 2003 and 2009 for AIS, 12-15 years old, Risser 0-3.Patients were divided in two groups, one of patients already treated with a brace (BRACE Group), and one never treated before (UNTREATED Group).The threshold of 5° Cobb to define worsened, improved and stabilized curves was considered, average Cobb angle, ATR, TRACE  Statistical analyses: Mean and SD were used for descriptive statistics of clinical and radiographic changes. Relative Risk of failure (RR), 95% Confidence Interval (CI), Student’s t, Kruskall Wallis, and chi square test were applied.268 patients were included , age 13.3 (±1).BRACE Group: 108 , age 13.2 (±1), Cobb Angle 34±12°, ATR 9.6±0.4, TRACE 5.7.UNTREATED Group: 160 , age 13.2 (±1), Cobb Angle 33±11° ATR 10.3±0.2°, TRACE 5.No differences among groups at first visit but for TRACE (p<0.05).49.38% of patients improved in UNTREATED, 43.13 stable, 7.50 worsened vs 35.19, 52.78 and 12.04 for BRACE (p=0.06). The Cobb angle was 28.9 vs 30.1 (p=0.06). The RR of failure for BRACE was 1.6 (IC95%0.86-2.35). No differences among groups for TRACE and ATR. Drop out had results similar to the completers (NS).Average clinical and radiological parameters improved in both groups. In the UNTREATED group results were slightly better even not significant, probably for the low statistical power. This study demonstrate that with a good treatment it’ possible to achieve good clinical results even in already treated patients.


Decreased activity and expression of the cardiac sarcoplasmic reticulum calcium ATPase (SERCA2a), a critical pump regulating calcium cycling in cardiomyocyte, are hallmarks of heart failure. We have previously described a role for the small ubiquitin-like modifier type 1 (SUMO-1) as a regulator of SERCA2a and have shown that gene transfer of SUMO-1 in rodents and large animal models of heart failure restores cardiac function. Here, we identify and characterize a small molecule, N106, which increases SUMOylation of SERCA2a. This compound directly activates the SUMO-activating enzyme, E1 ligase, and triggers intrinsic SUMOylation of SERCA2a. We identify a pocket on SUMO E1 likely to be responsible for N106's effect. N106 treatment increases contractile properties of cultured rat cardiomyocytes and significantly improves ventricular function in mice with heart failure. This first-in-class small-molecule activator targeting SERCA2a SUMOylation may serve as a potential therapeutic strategy for treatment of heart failure. SUMOylation of the cardiac calcium pump SERCA2a affects its activity and promotes cardiomyocyte contractility. Here the authors identify a small molecule N106 that increases SERCA2 SUMOylation and improves heart function in mice, and propose a promising therapeutic strategy for treatment of heart failure. Impaired calcium reuptake resulting from decreased expression and activity of SERCA2a is a hallmark of HF. The work in our laboratory has led to the successful completion of Phase 1 and Phase 2 clinical trials of adeno-associated vector type 1 (AAV1)-mediated gene transfer of SERCA2a in patients with severe HF, showing clinical benefits in patients receiving AAV1.SERCA2a34579There are 26 million people who suffer from heart failure (HF) in the world (5.8 million patients in the United States)SUMOylation, a ubiquitin-like reversible PTM where SUMO  covalently attaches to a target protein through specific enzyme cascades reaction, has been shown to be implicated in controlling many cellular processes, including regulation of protein function, stability and localization13N-(4-methoxybenzo[d]thiazol-2-yl)-5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-amine (molecular weight 354gpermol)), that enhances SERCA2a SUMOylation, resulting in enhanced contractility both in vitro and in vivo. In addition, we characterize the putative site on E1 ligase where N106 docks and confers its activity. Thus, our studies have established a compelling rationale for targeting SUMOylation of SERCA2a as a new therapeutic approach for the treatment of HF.In this report, we have conducted a screening study to discover small molecules hat increase SUMOylation. We identify a small molecule, N106 S-transferase (GST)-tagged form of SUMO-1 in the presence of E1, E2 (Ubc9) and ATP. Following the SUMOylation reaction, glutathione-coated donor beads and Ni2+-coated acceptor beads are added to interact with GST and His, respectively, effectively capturing the SUMOylated substrate. When the beads are brought into proximity by the SUMOylated protein, irradiation of the donor beads at 680 nm will result in emission of light at 520–620 nm. The Kd determined by FRET between SUMO-1 and RanGAP1 is compatible with those determined with other traditional approaches, such as isothermal titration calorimetry and surface plasmon resonance. We have used this technology to screen over 100,000 compounds from large NCI libraries and a few compounds have been shown to activate SUMOylation at a concentration of 10 μM. Among molecules that increased the RanGAP1 SUMOylation, we performed subsequent functional assays. A compound was considered a primary hit if it increased the FRET signal greater than threefold the s.d. above the mean of the vehicle controls when tested at 10 μM. Hits were subsequently filtered to remove all compounds containing substructural features common to promiscuous compoundsTo identify small-molecule activators of the SUMOylation pathway, we used the data from P<0.001, Student's t-test, n=25 cells per heart, three hearts were used) and reduced the rate of transient calcium decay (τ (s); N106 0.18±0.02, DMSO 0.24±0.02; P<0.05, Student's t-test), and significantly induced endogenous SERCA2a SUMOylation in isolated adult cardiomyocytes : 10 nM, −1.93±0.08, P<0.05, Student's t-test; 100 nM, −2.17±0.10, P<0.05, Student's t-test; and 10 μM, −2.30±0.19, P<0.001, Student's t-test); and relaxation  in a concentration-dependent manner : 10 nM, 0.20±0.01, P<0.05, Student's t-test; 100 nM, 0.20±0.01, P<0.05, Student's t-test; and 10 μM, 0.16±0.008, P<0.001, Student's t-test) was also significantly decreased with increasing amounts of N106, indicating faster SERCA2a-mediated Ca2+ uptake ; N106 12.03±0.80, dimethylsulfoxide (DMSO) 3.05±0.47; myocytes . The chet manner . Calcium+ uptake . Further+ uptake  and leve+ uptake . Whole S+ uptake . These dP<0.001, Student's t-test; 1 h, 8.60±0.23, P<0.001, Student's t-test; and 24 h, 8.90±0.38, P<0.001, Student's t-test; maximal velocities of cell contraction (μm s−1): 10 min, −1.97±0.04, P<0.05, Student's t-test;1 h, −1.92±0.09, P<0.05, Student's t-test; and 24 h, −2.3±0.19, P<0.001, Student's t-test; maximal velocities of cell relaxation (μm s−1): 10 min, 1.12±0.11, P<0.05, Student's t-test; 1 h, 1.67±0.15, P<0.05, Student's t-test; and 24 h, 1.99±0.15, P<0.001, Student's t-test; calcium transient : 10 min, 0.19±0.01, P<0.05, Student's t-test; 1 h, 0.17±0.01, P=0.17, Student's t-test; 24 h, 0.18±0.008, P<0.05, Student's t-test; t90 (s): 10 min, 0.40±0.03, P<0.05, Student's t-test; 1 h, 0.35±0.01, P<0.05, Student's t-test; and 24 h, 0.35±0.006, P<0.001, Student's t-test; τ (s): 10 min, 0.17±0.02, P<0.05, Student's t-test; 1 h, 0.14±0.02, P<0.001, Student's t-test; and 24 h, 0.16±0.008, P<0.001, Student's t-test), SERCA2a's ATPase activity and SUMOylation short amount of time (10∼30 min), and these effects were sustained at 24 h in adult cardiomyocytes (Time-course experiments showed that N106 increased cell contractility (peak shortening (%): 10 min, 9.65±0.27, myocytes .50=2.45±0.13 μM, in-vitro thioester formation between the SUMO E1 and SUMO-1 was ∼20 μM range. The SUMO-1 conjugation to SUMO E1 enzyme and SUMO E1-dependent thioester bond formation between SUMO E2 enzyme and SUMO-1 were less pronounced at 50 μM concentration and that was inhibited at over 100 μM concentration  and closed (the activating state) states during the SUMOylation processin-vitro activity of the ubiquitin-activating enzyme (Ub E1) at concentrations up to 100 μM, including ubiquitin activation, conjugation of ubiquitin to Ub E1 enzyme and Ub E1-dependent conjugation of ubiquitin to the E2 enzyme cdc32  or sham operation and received either N106 or vehicle. At 2 months, post TAC animals developed HF. The left ventricle (LV) was severely dilated and the cardiac function as determined by fractional shortening and ejection fraction, were significantly decreased . The SERτ, a parameter of relaxation, decreased, indicating enhanced relaxation (max in both sham and TAC mice (in-vivo system) and bioavailability of the N106 compound in vitro versus in vivo.We found a beneficial effect on haemodynamic parameters following the infusion of N106. The end systolic pressure–volume relationship (ESPVR) in the LV was significantly steeper when N106 was infused compared with baseline . More imlaxation . ChangesTAC mice . We showTAC mice . This maP=0.07, Student's t-test). SERCA2a's ATPase activity was somewhat higher in N106-treated TAC hearts than vehicle-treated hearts  form. SUMOylation of Sp1 is known to decrease its protein stabilityAtp2a2 (Serca2) gene (Serca2 KO)Serca2 gene excision, the mice displayed a substantial reduction in systolic and diastolic function  larger in the Serca2 KO mice compared with the WT control , +dP/dtmax , Pmax , τ  and heart rate  were not different in the presence of N106  was chosen. It improved contractility in a dose-dependent manner and calcium-transient property in isolated cardiomyocytes. In the pressure-overload-induced HF model, we found that N106 induced sustained reversal of contractility and was associated with an increased SERCA2a SUMOylation profile.We tested the specificity of this novel compound N106 for SERCA2a, using molecular, cellular and animal model approaches. N106 seems to increase SUMOylation on SERCA2a with a subset of substrates, although it targets SUMO E1 enzyme. We showed that the extent of SUMOylation changes of individual proteins could be different in response to N106 treatment . One posUBA2 gene expression was higher and directly correlated with lower survival ratemyc-dependent tumour growth in mice and there is a correlation between low SUMO E1 gene expression and longer metastasis-free survival rate of patients with myc-high breast cancershttp://dtp.cancer.gov/docs/compare/compare.html). The NCI-60 set includes leukemia, lymphomas and carcinomas of ovarian, renal, breast, prostate, colon, lung and central nervous system origin. The results of the screen validated that the N106 did not induce any tumour cell growth and proliferation including myc-dependent human breast cancer cell line MDA-MB-231  was 56% and 50%, and terminal elimination half-life (t1/2) was 19 min  purchased from Jackson Laboratories . Male rats aged 3 months and weighting 180∼250 g were obtained from Jackson Laboratories.−1 creatine , 2 mmol l−1 l-carnitine (Sigma-Aldrich), 5 mmol l1− taurine (Sigma-Aldrich), 100 IU ml1− penicillin (Life Technologies) and 100 μg ml1− streptomycin (Life Technologies). At 4 h after plating, the culture medium was changed and small molecules were added. As indicators of cardiac functions, measurement of intracellular calcium and cell shortening were performed. In brief, the cardiomyocytes were loaded with the calcium indicator Fura-2 and stimulated at different frequencies (0.1∼3.0 Hz) using an external stimulator , whereas a dual-excitation spectrofluorometer  was used to record fluorescence emissions used to calculate intracellular calcium concentration. Cell shortening was measured using video microscopy motion-detector system.Ventricular heart muscle cells were isolated from 180∼250 g male Sprague–Dawley rats as described−1) and xylaxine (5 mg kg−1), and ventilated with a tidal volume of 0.1 ml and a respiratory rate of 110 breaths per minute . A 2- to 3-mm longitudinal cut was made in the proximal portion of the sternum, allowing visualization of the aortic arch. The transverse aortic arch was ligated between the innominate and left common carotid arteries with an overlaying 27-gauge needle. The needle was immediately removed, leaving a discrete region of constriction. Animal sham group underwent a similar procedure without ligation. Only animals with fractional shortening < 50% were determined as being in HF and suitable for further study.B6C3F1 mice were acquired from Jackson Laboratories. Mice underwent TAC using a supraclavicular construction model as described15−1) for echocardiographic analysis. Transthoracic echocardiography was performed using a Vivid 7,000 equipped  with a H13L transducer (14 MHz). Two-dimensional and M-mode images were obtained in the short-axis view. The HR, LV end-diastolic internal diameter and LV end-systolic internal diameter were measured over the course of at least three repeated cardiac cycles. The fractional shortening and ejection fraction were then calculated.Mice were anaesthetized with intraperitoneal ketamine , etomidate (10 μg g−1) and morphine (1 μg g−1), and were then intubated via a tracheotomy and mechanically ventilated at 7 μl g−1 tidal volume and 120 respirations per minute. A small incision was made in the apex and a French Millar cathether connected a high-fidelity pressure transducer  inserted into the LV. Haemodynamic recordings were performed after 5∼10 min of stable HR. The rate of pressure rise (+dP/dt) and maximal pressure, the time constant of isovolumic relaxation and HR were recorded and analysed. To determine the relative effects of the admittance and conductance techniques to contractility change induced by N106 compound infusion, whole haemodynamic function was recorded at both baseline and after administration of N106 compound through the external jugular vein. N106 compound was solubilized in 10% DMSO (Sigma-Aldrich), 10% Tween-80 (Sigma-Aldrich) and saline for in-vivo experiments. N106 or vehicle solutions were continuously infused through the external jugular vein at a rate of 50 μl min−1 for 2 min in each dose. Whole haemodynamic functions of increasing dose of N106 of each mouse were recorded for ∼4 h. Each dose of N106 solution was infused after back to baseline of heart function.One day after echocardiography, we performed Protein extracts (20∼50 μg) were obtained from isolated cardiomyocytes, which was lysed in RIPA buffer  contained protease inhibitors  and 20 mM NEM (Sigma-Aldrich). For SERCA2a SUMOylation assays, cardiac protein extracts were immunoprecipitated with a SUMO-1 agarose resin  for overnight at 4 °C. The resins were washed with cold lysis buffer three times. The immunocomplexes were then resolved by SDS–PAGE and western blot analysis was performed using a SERCA2a-specific antibody. For general western blotting, cardiac proteins (10∼50 μg) were separated on SDS–PAGE gel, transferred to nitrocellulose membrane  and blotted with the following primary antibodies: SUMO-1 , Ubc9 , SAE2 , SERCA2a , PLN , PLN phosphorylation at serine-16 , RyR2 , NCX , glyceraldehyde 3-phosphate , GATA4  and Sp1 . Secondary antibodies anti-mouse IgG-HRP  and anti-rabbit IgG-HRP  were purchased from Sigma-Aldrich. The immunoreactive bands were visualized by a supersignal chemiluminescence detection reagent . Full scans of western blots are presented in Escherichia coli BL21 from a bicistronic constructs of GST-SAE2 and untagged SAE1. GST-fusion SAE2/SAE1 proteins were purified by affinity chromatography on glutathione-Sepharose beads as outlined by the manufacturer .Human SAE1 and SAE2 were amplified by PCR from complementary DNA obtained from OriGene Tech  and cloned into the GST expression vector, pGEX-6P-3 . Expression vectors coding for WT SAE1/SAE2 were constructed as describedin-vitro gel-shifting assays. ATP hydrolysis activity of E1 enzymes was measured by using pyrophosphate assay kit (Abcam Inc.). E1 (0.05 μM), 0.25 μM E2, 12.5 μM SUMO-1 and 1 mM ATP with N106 or DMSO were used for the assays. The E1-thioester formation was analysed in reaction mixtures containing 0.05 μM E1, 12.5 μM SUMO-1 and 1 mM ATP with N106 or DMSO. The E1-dependent E2-SUMO-1 thioester formation was assessed using reaction containing 0.05 μM E1, 0.25 μM E2, 12.5 μM SUMO-1 and 1 mM ATP with N106 or DMSO. Assays were incubated at 37 °C for 30 min and were mixed with non-reducing sample buffer, and analysed in non-reducing condition. Western blotting using antibodies against SAE2 and Ubc9 detected the SUMO conjugations. To determine the effect of N106 on ubiquitin-activating enzyme E1 activity, similar assays were performed (in-vitro assays was prepared in DMSO (Sigma-Aldrich).Biochemical activity of SUMO-specific E1 enzymes was examined by ATPase assay and erformed . Inorgang for 10 min at 4 °C, then the supernatants were collected and centrifuged at 12,000g for 15 min at 4 °C. The supernatants were transferred to ultracentrifuge tubes and centrifuged for 1 h at 140,000g at 4 °C . The pellets were resuspended in homogenization buffer to determine protein concentration and calcium-dependent ATPase activity assay. Protein concentration was measured with a 2-D Quant Kit . The ATPase activity were measured at 25 °C in 50 mM MOPS , 100 mM KCl (Boston Bioproducts), 5 mM MgCl2 (Boston Bioproducts), 1 mM EDTA (Boston Bioproducts) at pCa 5.0 by using a coupled enzyme system consisting of pyruvate kinase and lactate dehydrogenase, and monitoring the oxidation of the reduced form of NADH at 340 nM.SERCA2a ATPase activity was determined using a coupled enzymes assay including pyruvate kinase/lactate dehydrogenase/NADH-coupled reactions. Microsomes were enriched by differential density centrifugation. The cardiac cells and tissues were homogenized on ice in homogenization buffer (250 mM sucrose (Sigma-Aldrich), 5 mM HEPES pH7.0 (Sigma-Aldrich), 1 mM phenylmethyl sulfonyl fluoride (Sigma-Aldrich), protease inhibitor cocktail ). The homogenates were centrifuged at 1,000−1 intravenously and 10 mg kg−1 orally (PO). The bloods were corrected at 30 min, 1, 2 and 4 h after administration intravenously and at 30 min, 1, 2 and 4 h after treatment for oral administration studies. Plasma samples from these studies were extracted by plasma separator tube containing lithium heparin . Plasma concentrations of N106 were determined by HPLC analysis and pharmacokinetic parameters. Three mice for each time point were used.The male mice were received with a small molecule N106 at 10 mg kgP-values calculated using Student's t-test for the change from baseline before treating with compound or vehicle control. Statistical analysis was performed using GraphPad Prism software .Data represents mean value±s.e.m. How to cite this article: Kho, C. et al. Small-molecule activation of SERCA2a SUMOylation for the treatment of heart failure. Nat. Commun. 6:7229 doi: 10.1038/ncomms8229 (2015).Supplementary Figures 1-14


To determine the contents of B-vitamins in human milk in China, we analyzed 1778 human milk samples from the sample bank of the National High Technique R & D Program (863 Projects) which was a cross-sectional survey and covered 6419 human milk samples from healthy lactating mothers who were at different stages of lactation (0–330 days postpartum) in 11 provinces of China. The contents of free forms of six B-vitamins in these human milk samples were analyzed by using UPLC-MS/MS. The median concentrations of free form of 6 B-vitamins in colostrums, transitional milk, 15–180 d mature milk and 181-330 d mature milk were respectively as follows: thiamin 5.0 µg/L, 6.7 µg/L, 21.1 µg/L and 40.7 µg/L; riboflavin 29.3 µg/L, 40.6 µg/L, 33.6 µg/L and 29.6 µg/L; niacin 470.7 µg/L, 661.3 µg/L, 687.0 µg/L and 571.3 µg/L; vitamin B-6 4.6 µg/L, 16.1 µg/L, 62.7 µg/L and 80.7 µg/L; flavin adenine dinucleotide (FAD) 808.7 µg/L, 1162.8 µg/L, 1023.9 µg/L and 1057.2 µg/L; pantothenic acid 1770.9 µg/L, 2626.8 µg/L, 2213.0 µg/L and 1895.5 µg/L. The contents of 6 B-vitamins varied significantly among the different lactation stages and different areas . The present study indicated that the concentrations of B-vitamins in colostrum were generally much lower than those in transitional milk and mature milk. Further studies are warranted for their roles and significance on B-vitamins in colostrum in nutrition and metabolism of neonates. Human milk has been recommended as the optimal food for infants and met almost all the nutrient requirements for infants aged 0 to 6 months. Human milk is not only essential for infants’ growth and development, but also provides immunological protection for infants , 2. Geneet al .–16.13–166.3 μg/L . Some ofet al [In general, the contents of thiamin, riboflavin and FAD, and niacin in breast milk were lower in rural areas than in urban areas. Previous studies also found such difference , 23. Theet al  suggesteThe contents of niacin in human milk were higher in coastal area than in inland area. Niacin widely exists in fishes and seafood, such as mussel, crab meat, small shrimp, mackerel, mandarin fish and so on . The conIn fact, we have analyzed some minor vitamins in human milk such as nicotinic acid, pyridoxine and pyridoxamine. However, the contents of them were too low to be detected in part of samples so that we did not report these values individually in present paper. We gave the sum of pyridoxal, pyridoxine and pyridoxamine as the vitamin B-6 and the sum of nicotinamide and nicotinic acid as niacin. In our study, 4.3% mothers (n = 76) had taken nutrient supplements during lactatione. The major supplements included calcium tablet, protein, cubilose, and so on. Only five mothers received the vitamin supplements and they were excluded already.The limitation of the study was that the free forms of B-vitamins only were analyzed so that the findings can not reflect the total level for these vitamins including bound forms. For determination of bound forms of B-vitamins, e.g. thiamin monophosphate, pyridoxal-5’-phosphate, it should use more intensive pretreatment such as enzymolysis and acidolysis and different enzyme or different treatment condition for specific vitamin which would be a limitation for simultaneous analysis of free and bound vitamin forms. In subsequent analysis, we should consider how to analyze the bound forms of B-vitamins individually. Moreover, this study was a cross-sectional survey, which limits the possibility to study the changes of vitamin levels from the same subjects over the course of lactation as a longitudinal study would do.In summary, the contents of six B-vitamins  were lower in colostrums than in transitional and mature milk in China. The levels of B-vitamins were consistent with previous reports, but larger variations were present which could reflect the differences in breast milk collection measures and different analytical methods. Our findings can reflect the general level of these B-vitamins in human milk from apparently healthy lactating women in China. The contents of riboflavin, FAD, vitamin B-6 and pantothenic acid were affected by lactation stage, environment and region.S1 Table(XLSX)Click here for additional data file.S1 Fig(DOC)Click here for additional data file.


Rickettsia aeschlimannii is a tick-associated human pathogen. We report here the draft genome of R. aeschlimannii strain MC16, isolated from Hyalomma marginatum marginatum ticks collected in Morocco. Rickettsia spp. are strictly intracellular bacteria that are associated with arthropods. Some of them cause human infections that are currently considered emerging and have been identified worldwide  using a mate-pair library. Quality trimming and de novo assembly of the reads were performed using the CLC Genomics Workbench version 6.0.1 . The resulting contigs were reordered in Mauve version 2.3.1 (Rickettsia montanensis strain OSU 85-930 as a reference genome (accession no. CP003340.1). Potential coding sequences (CDSs) were predicted using AMIGene  inflation parameter of 1.5.The MiSeq sequencer  was used to sequence the genomic DNA of  AMIGene , GenBank AMIGene  database AMIGene , while r AMIGene , and RNA AMIGene , respectOrthoMCL , with a R. aeschlimannii MC16 consists of 16 contigs ranging in size from 314 to 238,303 bases, resulting in a total genome of 1,312,196 nucleotides, with an average genome coverage of 145-fold and a G+C content of 32.2%. Two plasmids of 14,948 bp and 26,886 bp, respectively, were identified and exhibited identity matches of 96%  to Rickettsia rhipicephali strain 3-7-female6-CWPP plasmid pMCC_1 (accession no. CP003343.1) and 96%  to Rickettsia monacensis strain IrR/Munich plasmid pRM (accession no. EF564599.1), respectively. The chromosome contains 1,790 CDSs and, like other rickettsial genomes, 3 noncontiguous rRNAs , 33 tRNAs, and 3 other RNAs. In addition, the two plasmids contain 23 and 44 CDSs but no RNAs.The draft genome of R. aeschlimannii chromosome exhibits a high level of synteny to R. montanensis but differs from it by three inversions of 66,949 bp, 3,404 bp, and 53,495 bp and the absence of the genes encoding the integration host factor beta-subunit (himD), large extracellular α-helical protein, 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase (fadB), and competence protein F (comF1 and comF2).The CCEA01000001 to CCEA01000014 (BioProject PRJEB6087), and the two plasmid sequences have been deposited under accession no. CCEB01000001 and CCEB01000002.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under accession no.