The dihedral angle between the benzene and pyrrole rings is 11.1 (2)°. In the crystal, molecules are linked  18H10Cl2N2O2; systematic name: 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolopyrrole-1,4-dione] was solved from laboratory X-ray powder diffraction data using the simulated annealing method followed by Rietveld refinement because the very low solubility of the pigment in all solvents impedes the growth of single crystals suitable for X-ray analysis. The molecule lies across an inversion center. The dihedral angle between the benzene ring and the pyrrole ring in the unique part of the molecule is 11.1 (2)°. In the crystal, molecules are linked via N—H⋯O hydrogen bonds, forming chains along [110] incorporating R22(8) rings.The crystal structure of Pigment Red 254 [P.R. 254, C Within the range of diketopyrrolo-pyrrole (DPP) pigments presently offered to the market, P.R. 254 plays the most important role ] is 11.1 (2)°. An intramolecular C—H⋯O hydrogen bond occurs  supplied by Clariant Produkte (Deutschland) GmbH was taken as is.TOPAS  was generated automatically by the DASH-to-TOPAS link.Crystal data, data collection and structure refinement details are summarized in Table 2et al., 2016DASH  was used to solve the crystal structure from the powder pattern in direct space. The starting molecular geometry was built from known crystal structure of similar compound from the Cambridge Structural Database  = 40.7  = 82.8 . The molecule has an inversion centre, which means the asymmetric unit must consist of a one half of the molecule.Accurate peak positions for indexing were obtained by fitting 20 manually selected peaks with an asymmetry-corrected Voigt function. Indexing was done with the program wpR = 13.57, exp R = 11.20, χ2 = 1.467.Pawley refinement . Anisotropic peak broadening was included to allow the peak profiles to be described accurately. The discrepancies between the observed and the calculated profile appeared to systematically depend on the 10.1107/S2056989017003309/lh5832sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989017003309/lh5832Isup2.rtvRietveld powder data: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017003309/lh5832Isup3.cmlSupporting information file. DOI: 1517793CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Escherichia coli strains has not been characterized in Indian population till date. In this data article, we have described important VFs of uropathogenic E. coli (UPEC) that is P fim, Type-1 fim, cell surface hydrophobicity, mannose resistant haemagglutination/mannose sensitive haemagglutination (MRHA/MSHA) expression and α-haemolysin production. The data includes a profile of the five VFs investigated in E. coli isolates from sepsis patients (N=78) and control group (N=50) from non-sepsis subjects. We found that P fim phenotype was expressed in 25.3% of E. coli isolates from sepsis patients, whereas Type-1 fimbriae was detected in 30.5%. Cell surface hydrophobicity phenotype was present in 30.5%, α-haemolysin in 26.3% and MRHA/MSHA in 22.1% of sepsis E. coli isolates. None of the control E. coli isolates showed presence of these phenotypes. The combined phenotypic profile of all the five VFs was significantly higher in sepsis patients as compared to the control group.Bacterial virulence factors (VFs) influence the site and severity of urinary tract infections (UTI) and further leading to sepsis infection. Phenotypic characterisation of VFs specific to sepsis  Cell surface hydrophobicity phenotype was present in 30.52% of E. coli isolates whereas 26.31% were expressing α-haemolysin and MRHA/MSHA phenotype was shown by 22.1% of E. coli sepsis isolates (E. coli isolates as compared to the control group (p<0.01). Further combined expression profile of five phenotype virulence factors was significantly higher in sepsis E. coli isolates as compared to control group (p<0.001) (The phenotypic profiling of important virulence factors (VFs) have shown that P fim phenotype was expressed in 25.26% of tination A. The exisolates A. Similap<0.001) B.Fig. 1 were obtained from the stock library of Department of Microbiology, Vardhman Mahavir Medical College and Safdarjung hospital, New Delhi, India. The E. coli strains were collected from confirmed sepsis patients who visited the hospital while control group consists of the faecal E. coli isolates from non-sepsis controls. The bacteria were grown on tryptic soy agar (TSA) agar plates at 37 °C overnight and further stored at 4 °C for the phenotypic characterisation.2.2D-mannoside. Strains causing mannose-sensitive agglutination were defined as Type 1 fimbriated The phenotype of P-fimbrial was defined by P blood group dependent haemagglutination 2.3+ve, 3% v/v in 1× PBS) and one drop of PBS, with or without D-mannose (3% w/v). The plate was left to rotate (15 rpm) for 5 min at 25 °C followed by rotation for 5 min at 4 °C. Haemagglutination was considered to be mannose-resistant (MRHA) when it occurred in the presence of mannose and mannose-sensitive (MSHA) when it was inhibited by mannose Haemagglutination was performed in round-bottomed microtitration plates. One drop (100 µl) of bacterial suspension was mixed with one drop of erythrocytes (human A2.49 cfu/ml) in 0.2 M phosphate buffer, pH 6.8, of bacteria grown on TSA medium. In brief, suspensions were mixed with ammonium sulphate solutions at final molar (M) concentrations of 2.0, 1.4, 1.0, 0.4, 0.1, 0.06 and 0.02. Strains were considered to be hydrophobic when they aggregated in ammonium sulphate at concentrations ≤1.4 M The cell-surface hydrophobicity was calculated by the salt aggregation test (SAT) with suspensions (5×102.5Sheep blood agar plates were used for determination of α-haemolysin production that contained 1% sheep blood (v/v). About 7–8 wells of 8 mm diameter were made on blood agar plate and 50 µl of bacterial lysate was poured into wells and incubated overnight. Zone of inhibition was recorded. Strains with a clear halo after overnight culture at 37 °C were defined as haemolytic 3P values≤0.05 were considered as statistically significant.The chi-square test was used for statistical comparison between the two groups.


Metabolic reprogramming, a crucial cancer hallmark, shifts metabolic pathways such as glycolysis, tricarboxylic acid cycle or lipogenesis, to enable the growth characteristics of cancer cells. Here, we provide evidence that transketolase-like 1 (TKTL1) orchestrates aerobic glycolysis, fatty acid and nucleic acid synthesis, glutamine metabolism, protection against oxidative stress and cell proliferation. Furthermore, silencing of TKTL1 reduced the levels of sphingolipids such as lactosylceramide  and phosphatidylinositol . Thus, in addition to its well-known roles in glucose and amino acid metabolism, TKTL1 also regulates lipid metabolism. In conclusion, our study provides unprecedented evidence that TKTL1 plays central roles in major metabolic processes subject to reprogramming in cancer cells and thus identifies TKTL1 as a promising target for new anti-cancer therapies. To testy Figure , but shoy Figure  since phKD cells, which perfectly fits with theoretical predictions for the one-substrate reaction model  in THP-1l Figure . In HCT1l Figure .KD) cells. SiRNA-mediated silencing reduced ACLY transcript levels by 45 ± 10%  without affecting proliferation, glucose consumption or lactate production  is a key enzyme regulating lipid synthesis by converting cytosolic citrate to AcCoA. To evaluate the possible role of TKTL1 in producing AcCoA in comparison with ACLY, we silenced ACLY in HCT116 . TRIzol™ reagent was obtained from Invitrogen . RNase was obtained from Roche Diagnostics . Phospholipid and sphingolipid standards were purchased from Sigma-Aldrich Co.   or Avanti Polar Lipids, Inc.  (Cer 24:1 and sphinganine 17:0).All chemicals were purchased from Sigma-Aldrich Co , unless otherwise specified. Antibiotics  were obtained from Gibco-BRL , fetal calf serum (FCS) and trypsin-EDTA solution C (0.05% trypsin - 0.02% EDTA) from PAA Laboratories . Stable -D-glucose (Isotec) was analyzed by GC-MS as described -glucose (Sigma-Aldrich) (50% isotope enrichment), for 48 h. Following incubation, cells were counted with a Scepter Handheld Automated Cell Counter (Millipore) and centrifuged . Medium was collected for glucose, lactate and glutamate/glutamine analysis, and cell pellets were frozen for RNA ribose and fatty acids analysis.For isotopologue distribution analysis, 3 × 10Glucose was isolated from 200 μL medium by ion-exchange chromatography and converted to its aldonitrile acetate derivative by adding 100 μL hydroxylamine in pyridine (2% wt) at 100°C for 30 min followed by 75 μL acetic anhydride at 100°C for 1 h. The reaction mixture was evaporated to complete dryness and redissolved in 50 to 200 μL of ethyl acetate. 1 μL of the derivatized product was loaded to a GC-2010 gas chromatograph (Shimadzu) equipped with a HP-5MS capillary column , connected to a GCMSQP2010 mass selective detector (Shimadzu) [Lactate was extracted from the medium by ethyl acetate after acidification with HCl and 1 mL ethyl acetate at 20°C. The propylamideheptafluorobutyric derivative of the lactate was prepared and analyzed as described [RNA ribose was isolated by acid hydrolysis of cellular RNA after Trizol (Invitrogen) extraction from cell pellets. Ribose was derivatized to its aldonitrile acetate form by an analogous derivatization procedure as described for glucose. After derivatization, the reaction mixture was evaporated to complete dryness and redissolved in 50 to 200 μL of ethyl acetate. Glutamate was isolated from 1 mL of cell medium using ion-exchange chromatography and converted to its n-trifluoroacetyl-n-butyl derivative by adding 200 μL of butanolic HCl (0.4 N), the mixture incubated at 100°C for 1 h and dried under nitrogen stream. Next, 100 μL methylene chloride and 25 μL trifluoroacetic acid were added. The solution was kept at 20°C for 20 min and dried under nitrogen stream. The pellet was dissolved in 50 to 200 μL methylene chloride and 1 μL was injected for analysis [Fatty acids were extracted with methanol from the organic phase obtained during RNA isolation with Trizol (Invitrogen). Purified fatty acids were converted to their methyl ester derivatives by adding 200 to 500 μL of methanolic HCl 0.5 N, heating at 70°C for 1 h, drying under nitrogen stream and dissolving in 200 μL of hexane for injecting 1 μL in the GC-MS, now equipped with a bpx70 (SGE) column .himadzu) . Lactateescribed . RNA ribanalysis , 55. Fat3 cells each were re-suspended in 100 μL of milliQ water and 500 μL pure methanol (MeOH), these suspensions were transferred to glass vials. 250 μL chloroform (CHCl3) was added to obtain an extraction solvent MeOH:CHCl3:H2O (2:1:0.4). 10 μL of PL or SL standard mix was added and lipids were extracted overnight at 48°C. For sphingolipid analysis, 75 μL of 1 M potassium hydroxide (KOH) dissolved in MeOH was added and samples were sonicated. After 2 h of incubation at 37°C, 75 μL of 1 M acetic acid was added. Following extraction, PL and SL samples were dried under nitrogen stream and stored at −20°C until further usage. Prior to analysis on an UPLC-MS, samples were dissolved in 500 μL MeOH and transferred to Eppendorf tubes. The volume was reduced by evaporation under gentle nitrogen steam to a final volume of 150 μL MeOH, prior to analysis samples were centrifuged at 10,000 x g for 3 min and 150 μL of supernatant was infused into the UPLC. The liquid chromatography–mass spectrometer  was operated in positive electrospray ionisation mode. Full scan spectra from 50 to 1,500 Da were acquired and individual spectra were summed to produce data points every 0.2 s. Mass accuracy and reproducibility were maintained by using an independent reference spray via LockSpray interference. The analytical column, maintained at 30°C, was a 100 × 2.1 mm id, 1.7 μm C8 Acquity UPLC BEH (Waters). The mobile phase consisted of solvent A: MeOH/H2O/HCOOH (74:25:1 v:v:v); and solvent B: MeOH/HCOOH (99:1 v:v), both containing 5 mM ammonium formate. A gradient was programmed - 0.0 min, 80% B; 3 min, 90% B; 6 min, 90% B; 15 min, 99% B; 18 min, 99% B; 20 min, 80% B; 22 min, 80% B, with a 300 μL/min flow rate. Quantification was carried out using the ion chromatogram extracted for each compound in 50 mDa windows. The linear dynamic range was determined by injecting standard mixtures. Positive identification of compounds was based on the accurate mass measurement with an error < 5 ppm and its LC retention time, compared to that of a standard (± 2%). Lipid species were assigned from the m/z values detected in the MS using the MassLynx software (v4.1 SCN627 Waters). Positive ionization allowed detection of PC, PC-pmg, Lyso-PC and Lyso-PC-pmg from the phospholipid extract and Cer, dhihydroceramides, SM, dihydrosphingomyelins, GlcCer, glucosyldihydroceramides, LacCer and lactosyldihydroceramides from the sphingolipid extract. Negative ionization mode allowed detecting the rest of phospholipid classes: PE, PE-pmg, Lyso-PE, Lyso-PE-pmg, PS, Lyso-PS and PI.Two standard mixes of phospholipids (PL) and sphingolipids (SL) were prepared. The PL mix contained PC 34:0, PE 34:0, PG 34:0, PI 34:2, PS 34:0 and LysoPC 17:0 at a concentration of 20 μM each. The SL mixture contained Cer 12:0, GlcCer 12:0, LacCer 12:0, SM 12:0 and sphinganine 17:0 at a concentration of 20 μM each. These mixes were used as internal standards for PL and SL identification and quantification at a final concentration of 200 pmol/sample. Two pellets containing 750 × 10In vitro experiments were performed in triplicate for each treatment in at least two independent experiments. Mass spectrometric analyses were carried out in three independent 1 μL injections of each sample by the automatic sampler in a linear set-up. Raw data were considered valid only if the sample deviation was < 1% of the normalized peak intensity. Statistical analyses were performed using the parametric unpaired, two-tailed independent sample Student's t-test, assuming normality of distribution and homogeneity of variance based on past experience in similar measurements [P < 0.05 (*) was considered statistically significant.urements , 57. Wheurements . P < 0.0


Our experiments indicate that these complexes have ionophoric properties and redox activity. They appeared to be very effective generating reactive oxygen species and deregulating the antioxidative potential of a cell. Moreover, the genes that are responsible for antioxidant capacity were considerably deregulated, which led to the induction of apoptosis and cell cycle arrest. On the other hand, good intercalating properties of the studied compounds may explain their ability to cleave DNA strands and to also poison related enzymes through the formation of reactive oxygen species. These findings may help to explain the particularly high selectivity that they have over normal cells, which generally have a stronger redox equilibrium.Thiosemicarbazones are chelators of transition metals such as iron or copper whose anticancer potency is intensively investigated. Although two compounds from this class have entered clinical trials, their precise mechanism of action is still unknown. Recent studies have suggested the mobilization of the iron ions from a cell, as well as the inhibition of ribonucleotide reductase, and the formation of reactive oxygen species. The complexity and vague nature of this mechanism not only impedes a more rational design of novel compounds, but also the further development of those that are highly active that are already in the preclinical phase. In the current work, a series of highly active thiosemicarbazones was studied for their antiproliferative activity  The disruption of the cellular redox homeostasis of tumor cells appears to be an attractive and promising approach for cancer therapy. Cancer cells have increased reactive oxygen species (ROS) levels compared to normal cells. This phenomenon is associated with oncogenic transformations and glycolytic metabolic adaptations, which leads to the acceleration of metabolism . Thus, t+/NADH, NADP+/NADPH and GSH/GSSG, which characterize the cellular redox state piperazine (5 mmol) in methylene chloride (25 mL) were stirred for 24 h at room temperature. The solutions were extracted three times with distilled water and the organic phases were dried over MgSO4 and evaporated. The obtained thioketones were refluxed for 2 hours with hydrazine hydrate (5 mmol). Finally, crude thiosemicarbazides were crystallized from dry methanol to obtain pure crystals. NMR spectra and mass spectrometry analysis is provided in The mixtures of  bis-11H-NMR : δ 3.09 , 3.87 , 4.77 , 6.97 , 7.05 , 9.19 . 13C-NMR : δ 40.3; 49.1; 115.9; 117.9; 148.0; 157.8; 183.0.4-(4-fluorophenyl)piperazine-1-carbothiohydrazide Light pink powder; yield 97%; mp:180–181° C; 1H-NMR : δ 3.51 , 3.88 , 4.83 , 8.20 , 8.56 , 9.18 . 13C-NMR : δ 47.3; 48.1; 120.2; 122.8; 125.0; 136.8; 143.5; 159.7; 183.2.4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]piperazine-1-carbothiohydrazide White powder; yield 85%; mp:191–192° C; Two drops of glacial acetic acid were added as a catalyst to the mixtures of thiosemicarbazides (0.5 mmol) and appropriate quinolinecarboxaldehyde (0.5 mmol) in ethanol (5 mL). The glass tubes were sealed and placed into a microwave reactor at 83° C for 20 minutes (the reactor power did not exceed 50 W). The final products were crystallized from methanol.N’-[(quinolin-2-yl)methylidene]piperazine-1-carbothiohydrazide (1b) Yellow powder; yield 62%; mp:192–193° C; 1H-NMR : δ 3.26 ; 4.13 ; 7.02 ; 7.09 ; 7.63 ; 7.78 ; 7.99 ; 8.03 ; 8.35 ; 8.39 ; 11.67 . 13C-NMR : δ 49.4; 50.4; 115.8; 115.9; 117.8; 127.6; 128.2; 128.5; 129.3; 130.5; 137.2; 144.4; 147.9; 154.3; 155.8; 157.6; 181.2. HRMS-ESI: calcd for C22H21FN4S 394.1502 [M+H]+ found 394.1494.4-(4-fluorophenyl)-N’-[quinolin-2-yl)methylidene]piperazine-1-carbothiohydrazide (1c) Yellow powder; yield 98%; mp:126–127° C; 1H-NMR : δ 3.69 ; 4.14 ; 7.62 ; 7.79 ; 8.01 ; 8.23 ; 8.34 ; 8.38 ; 8.59 ; 11.68 . 13C-NMR : δ 19.0; 48.3; 50.2; 56.5; 117.8; 120.0; 127.6; 128.2; 128.5; 129.3; 130.5; 136.9; 137.1; 143.5; 144.4; 147.9; 154.3; 159.6; 181.6. HRMS-ESI: calcd for C22H19ClF3N5S 479.1033 [M+H]+ found 479.1025.4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]-2 flasks (Nunc). DMEM for HCT116 and MCF-7 were supplemented with 12% heat-inactivated fetal bovine serum (Sigma) and for NHDF with 15% non-inactivated fetal bovine serum (Sigma). Cells were cultured under standard conditions at 37° C in a humidified atmosphere at 5% CO2.The human colon cancer cell line HCT116 wild type and human breast carcinoma cell line MCF-7 were obtained from ATCC and the normal human fibroblast cell lines NHDF were obtained from PromoCell. Cells were grown as monolayer cultures in Dulbecco’s modified Eagle’s medium with an antibiotic gentamicin (200 μL/100 mL medium) in 75 cm® AQueous One Solution-MTS (Promega) was added to each well (with 100 µL DMEM without phenol red) and incubated for 1 h at 37° C. The optical densities of the samples were analyzed at 490 nm using a multi-plate reader . Results were expressed as a percentage of the control and calculated as the inhibitory concentration (IC50) values (using GraphPad Prism 5). The IC50 parameter was defined as the compound concentration that was necessary to reduce the proliferation of cells to 50% of the untreated control. Each individual compound was tested in triplicate in a single experiment with each experiment being repeated three or four times.The cells were seeded in 96-well plates (Nunc) at a density of 3,500 cells/well (HCT116 and MCF-7) and 3,000 cells/well (NHDF) and incubated at 37° C for 24 h. The assay was performed following a 96 h incubation with varying concentrations of the compounds that were being tested. Then, 20 µL of CellTiter 964, FeSO4 or FeCl3 was added into wells with the tested compounds. The results were expressed as a percentage of the control and calculated as the inhibitory concentration (IC50) values using GraphPad Prism 5. Each individual compound was tested in triplicate in a single experiment with each experiment being repeated three times.The HCT116 and MCF-7 cells were seeded in 96-well plates (Nunc) at a density of 3,500 cells/well and incubated at 37° C for 24 h. The MTS assay was performed following a 96 h incubation with varying concentrations of the tested compounds. Additionally, a 20 µM solution of CuSO5 cells/well and incubated at 37° C. After 24 h, freshly prepared solutions of the tested compounds: 1b, 1c, 2a-b, 3a-c and DOX (two-fold IC50 concentration) were added. The next day, the solutions of the tested compounds were removed and the cells were washed with Phosphate Buffered Saline (PBS), after which 5 μM CellROX® Green Reagent (Molecular Probes™) was added. After 30 min of incubation at 37° C, the cells were washed with PBS and then DMEM without phenol red was added. The observation was performed using an inverted fluorescence microscope  equipped with a CO2 incubator using a 485 nm excitation laser and a 520 nm emission filter.The HCT116 cells were seeded into eight-well chambers (Lab-Tek) at a density of 0.5 × 10® Green Reagent (Molecular Probes™). Additionally, the quantity of cells in each well was determined using Hoechst 33342 (Molecular Probes™). The solutions of the tested compounds were removed and 100 µL of CellROX Green Reagent and Hoechst 33342 at a final concentration of 5 μM were added to each well. Then, the cells were incubated for 30 min at 37° C. The fluorescence was measured using a multi-plate reader  at 485 nm excitation and a 520 nm emission for CellROX Green Reagent and a 345 nm excitation laser and a 485 nm emission filter for Hoechst 33342. The experiments were performed three to four times. ROS levels were expressed as the percentage of the control cells level.To determine the intracellular levels of ROS, HCT116 cells were seeded onto black 96-well plates (Corning) at a density of 9,000 cells/well and incubated at 37° C. After an overnight incubation, the solutions of the tested compounds: 1a-c, 2 a-b, 3 a-c, Dp44mT (1 μM) and DOX (5 μM) were added and incubated for 3, 6, 9, 12, and 24 h in a kinetic experiment. The generation of ROS was measured using a CellROX6 cells/well and incubated at 37° C. The next day, solutions of TSC (1 μM) and DOX (5 μM) were added and incubated for 3, 6, 9, 12, and 24 h in a kinetic experiment. The levels of intracellular GSH were measured using an enzymatic recycling method [To determine the intracellular level of reduced glutathione, the HCT116 cells were seeded in 3 cm Petri dishes (Nunc) at a density of 0.5 × 10g method . Briefly6 cells/well and incubated overnight. Next, the medium was removed and solutions of TSC (1 μM) and DOX (5 μM) were added. After 12 or 24 h, total RNA was isolated from the cells using TRIzol Reagent (Ambion) according to the manufacturer’s instructions. cDNA synthesis was performed with 5 μg of total RNA using a GoScript™ Reverse Transcriptase kit (Promega) and Oligo(dT)23 Primers (Sigma). The Real-Time PCR was performed with a CTX96 Touch™ Real-Time PCR Detection System (Biorad) in a 20 μL reaction volume. The reaction consisted of PowerUp™ SYBR® Green Master Mix (Applied Biosystems), a primer pair mix (0.5 μM each) and 1 μL of cDNA. All primer pair sequences were purchased from Sigma-Aldrich and are listed in –ΔΔCT method. The experiments were performed at least three times.The HCT116 cells were seeded in 3 cm Petri dishes (Nunc) at a density of 0.5·106 cells/well and incubated overnight. The next day, solutions of TSC (1 μM) and DOX (5 μM) were added and the cells incubated for 24 or 48 h. Cells were harvested by trypsinization and washed with cold PBS. Next, the cells were centrifuged and suspended in an RIPA buffer (Thermo Scientific) containing Halt Protease Inhibitor Cocktail (Thermo Scientific), Halt Phosphatase Inhibitor Cocktail (Thermo Scientific) along with 0.5 M EDTA and lysed for 20 min on ice. Then, the lysates were sonicated, centrifuged at 10,000 rpm for 10 min at 4° C and the supernatants were collected for further analysis. The protein concentration was determined using a Micro BCA™ Protein Assay Kit (Thermo Scientific) according to the manufacturer’s instructions. Equal amounts of the proteins (20 μg) were electrophoresed on SDS-Page gels and transferred onto a nitrocellulose membranes. The membranes were blocked in 5% non-fat milk prepared in PBS containing 0.1% Tween-20 (TPBS) for 1 h. After blocking, the membranes were incubated with specific primary antibodies: cdc2 , p21Waf1/Cip1 , p53  and β-Actin  overnight at 4° C, then washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1h at room temperature. All of antibodies were diluted 1:1000 in 5% milk in TPBS. Finally, the membranes were washed and incubated with a SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific). The chemiluminescence signals were captured using a ChemiDoc™ XRS+ System (BioRad). The experiments were performed at least three times. Densitometric analysis was performed using ImageJ 1.41 software .The HCT116 cells were seeded in 3 cm Petri dishes (Nunc) at a density of 0.5·106 cells/well and incubated at 37° C for 24 h. Then, the medium was removed and freshly prepared solutions of the tested TSC (0.5 μM) and DOX (5 μM) were added. After a 48 h treatment, assays were performed using a Muse Cell-Cycle Kit (Millipore) according to the manufacturer’s instructions. Briefly, cells were collected, washed with cold PBS and centrifuged at 300 g. Then, the cells were fixed in ice cold 70% ethanol and stored at –20° C overnight. Afterwards, the cells were centrifuged and resuspended in 200 μL of Muse™ Cell Cycle Reagent and incubated for 30 min at room temperature in the dark. After staining, the cells were processed for cell cycle analysis using a Muse Cell Analyzer (Millipore). The experiments were performed at least three times.The HCT116 cells were seeded in 3 cm Petri dishes (Nunc) at a density of 0.25·106 cells/well and incubated at 37° C for 24 h. Then, the medium was removed and freshly prepared solutions of the tested TSC (0.5 μM) and DOX (5 μM) were added. After 48 h, assays were performed using an Annexin V & Dead Cell Kit (Millipore) according to the manufacturer’s instructions. Briefly, detached and adherent cells were collected and centrifuged at 500 g for 5 min. Afterwards, the resuspended cells were incubated with 100 μL of Muse™ Annexin V & Dead Cell Reagent for 20 min at room temperature in the dark. After staining, the events for live, early and late apoptotic cells were counted using a Muse Cell Analyzer (Millipore). The experiments were performed at least three times.The HCT116 cells were seeded in 3 cm Petri dishes (Nunc) at a density of 0.25·10–1cm–1. The tested TSC and DOX were dissolved in DMSO to a concentration of 8.35 mM, which was then used as the stock solution for preparing the various concentrations  in 1 mL in 10 mM of Tris-HCl (pH 7.9). Afterwards, 18 µM CT-DNA was added to the prepared solutions, which were incubated for 1.5 h at 37° C with occasional vortexing. The absorption spectra were measured using a Hitachi U-2900 spectrophotometer in range of 200–500 nm. All absorption spectra were imported and compared in OriginPro 8.0.For the DNA binding studies, Calf-thymus DNA (CT-DNA) was purchased from Sigma Aldrich. The lyophilized CT-DNA was dissolved in 10 mM Tris-HCl, pH 7.9, mixed gently and left overnight at 4° C. The purity of the CT-DNA solution was determined by measuring the ratio of UV absorbance at 260 and 280 nm. A ratio more than 1.8 indicated that the DNA was sufficiently free from proteins. Then, the concentration of CT-DNA was determined from the absorbance at 260 nm using an extinction coefficient of 6600 Mt-test. Statistical differences in the expression of proteins, progression of cell cycle and Annexin V binding assay were calculated using one-way ANOVA with a Bonferroni post-hoc test. A p-value of 0.05 or less was considered to be statistically significant. GraphPad Prism v.5.0 software  was used for analysis.Results are expressed as the mean ± standard deviation (SD) from at least three independent experiments. Statistical analysis in the ROS, GSH measurements and expression of genes were performed using the two-tailed Student’s


To report 3-year results investigating the safety and efficacy of canaloplasty for open-angle glaucoma.  University of Oklahoma, Dean McGee Eye Institute, Oklahoma, United States of America.  Nonrandomized single-center retrospective chart review.  Adult open-angle glaucoma eyes underwent canaloplasty or combined cataract-canaloplasty surgery. A tensioning suture was placed into Schlemm's canal in all eyes. Primary endpoints included the mean IOP and mean number of glaucoma medications at each follow-up visit. Secondary endpoints included visual acuity and surgical/postsurgical complications. p < 0.001). The average medicine use was reduced from 2.1 preoperatively to 0.4 at 12 months, and 0.5 and 0.6 at two and three years, respectively (p < 0.001). The frequency of surgical and postsurgical complications was low with no serious adverse events recorded.  The study cohort included 277 eyes . Overall, the mean baseline IOP of 19.7 mmHg was reduced to 14.3 mmHg,14.0 mmHg, and 15.2 mmHg at 1, 2, and 3 years, respectively ( Canaloplasty was safe and effective in achieving long-term IOP reductions and reduced dependence on antiglaucoma medications. Until relatively recently, treatment for primary open-angle glaucoma (POAG) was restricted to medical therapy or traditional glaucoma surgeries such as drainage shunts and trabeculectomy, or laser trabeculoplasty. Trabeculectomy has a well-established IOP-lowering effect; however, it is also associated with numerous immediate and delayed postoperative complications, many of which stem from the creation of a subconjunctival bleb –6. FurthCanaloplasty is a nonperforating, blebless technique, eliminating many of the complications frequently seen with trabeculectomy. Even those complications that are associated with the procedure are usually transient and resolve quickly . This isPrior studies have reported on 1-, 2-, and 3-year results from prospective clinical trials of canaloplasty, which showed significant reductions in IOP and glaucoma medication usage in conjunction with an excellent safety profile –17. The This paper presents the 3-year results of a single-center retrospective chart review evaluating the intervention of canaloplasty at the University of Oklahoma, Dean McGee Eye Institute, performed by a single surgeon (MK) between January 2010 and December 2012. The cohort included 277 eyes affected by open-angle glaucoma. The study was performed in accordance with the principles stated in the Declaration of Helsinki. The current investigation was designed to demonstrate the safety and efficacy of canaloplasty in reducing intraocular pressure (IOP) and glaucoma medication dependence in the treatment of open-angle glaucoma (OAG). The protocol was approved by the institutional review board (IRB) at the University of Oklahoma.All patients provided informed consent. All enrollees had a complete baseline ophthalmic examination prior to surgery that included a history of glaucoma, medication use, IOP, best-corrected visual acuity (BCVA), gonioscopy, slit lamp, and fundus examination. Postoperative follow-up examinations were at 1 day, 1 week, and 1, 3, 6, 12, 18, 24, and 36 months. All relevant information was recorded at each follow-up visit, including IOP measurements, BCVA, slit lamp examination, gonioscopy, ophthalmic medications, and adverse events. Primary endpoints included the mean IOP and mean number of glaucoma medications at each follow-up visit. Secondary endpoints included surgical/postsurgical complications. Visual acuity codes such as count fingers (CF), hand motion (HM), and light perception (LP) are converted to LogMAR following Lee et al. [All patients were at a minimum 18 years of age at the time of enrollment, able to provide informed consent, and were scheduled for glaucoma surgery or combined cataract and glaucoma surgery. Inclusion criteria for this study included a glaucoma diagnosis of primary open-angle glaucoma (POAG), pigmentary glaucoma, or exfoliative glaucoma. Patients with advanced glaucoma exhibiting visual field loss were not excluded from the study. Patients with baseline IOP < 16 mmHg were not excluded from the study, and none of the patients had the utilization of antifibrotics. Patients with more than two laser trabeculoplasty (LTP) procedures were excluded. Other exclusion criteria included neovascular disease, uveitis, chronic angle closure glaucoma, angle recession, and developmental or secondary glaucoma. The protocol allowed for previous surgeries that would not interfere with complete circumferential catheterization of Schlemm's canal.Canaloplasty was performed using an iTrack™ microcatheter to circumferentially viscodilate and intubate Schlemm's canal with a tensioning suture, as previously described .t-tests or Wilcoxon's signed-rank tests, as appropriate. Two-tailed p values less than 0.05 were considered statistically significant. SAS  was used for the analysis of the data.The primary endpoints included the IOP and number of antiglaucoma medications at 1 day, 1 week, and 1, 3, 6, 12, 18, 24, and 36 months. The secondary endpoints included visual acuity and surgical/postsurgical complications. Descriptive statistics  were used to summarize the data. Comparisons of IOP, number of medications, and visual acuity were made against baseline at each time point via paired The study cohort consisted of 277 eyes with a mean age of 72.8 ± 10.9 years who met inclusion and exclusion criteria, provided consent for long-term follow-up, and completed baseline visits. p < 0.001). Similarly, the medication use was significantly decreased at all postoperative time points compared to baseline (p < 0.001). For all eyes, the mean baseline IOP of 19.7 ± 6.7 mmHg was reduced to 14.3 ± 4.6 mmHg at 12 months, 14.0 ± 4.2 mmHg at 24 months, and 15.2 ± 4.3 mmHg at 36 months (p < 0.001). The mean medicine use was reduced from 2.1 ± 1.2 before surgery to 0.4 ± 0.8 at 12 months, 0.5 ± 0.9 at 24 months, and 0.6 ± 0.9 at 36 months (p < 0.001) (< 0.001) .p < 0.001) (p < 0.001)  . Medicat< 0.001) .p < 0.001 for 12 and 24 months, p = 0.002 for 36 months) (p < 0.001).A statistically significant reduction in IOP was also observed in the 127 eyes undergoing phacocanaloplasty from a mean of 18.1 ± 5.6 mmHg at baseline to 14.4 ± 4.6 mmHg, 14.7 ± 4.4 mmHg, and 15.4 ± 4.0 mmHg at 12, 24, and 36 months, respectively ( months) . Mean meThe percentage of eyes with hyphema at a given time point is presented in p < 0.0001) at 1 day, 0.57 ± 0.42 with a Snellen fraction of 20/74.8 (p < 0.0001) at 1 week, and 0.40 ± 0.35 with a Snellen fraction of 20/49.8 (p < 0.0001) at 1 month (p < 0.05). At 18 months, 172 eyes had a visual acuity of 0.23 ± 0.36 with a Snellen fraction of 20/33.8 (p = 0.06). Two years after surgery, 143 eyes had a visual acuity of 0.22 ± 0.29 with a Snellen fraction of 20/33.3 (p < 0.001). At 3 years, visual acuity was not significantly better or worse, found to be 0.20 ± 0.24 with a Snellen fraction of 20/31.5 (p = 0.29) among 65 eyes  was 0.31 ± 0.35 with a mean Snellen fraction of 20/41.1. Visual acuities were found to be significantly worse at 1 day, 1 week, and 1 month following the procedure. Post-op visual acuity was found to be 0.85 ± 0.53 with a Snellen fraction of 20/140.2 ( 1 month . The pos 65 eyes .n = 150) and in n = 127), along with corresponding means in Snellen fractions and paired t-test results (versus pre-op VA in LogMAR). Rows in bold represent significantly worse visual acuity compared to pre-op, whereas rows in italic represent improved visual acuity. Broadly speaking, visual acuity worsens significantly after surgery but may return to preoperative values by around 3–12 months postoperatively.Mean and SD for visual acuity (LogMAR) are presented in IOP reduction is the mainstay of glaucoma treatment. The 3-year results reported here showed significant and sustained pressure lowering accompanied by a low incidence of postoperative complications. The reductions in IOP achieved over the long-term follow-up period are in line with previously reported studies in the literature and notably the landmark multicenter prospective trial carried out at 15 clinical sites in the United States, Great Britain, and Germany in 2005 .This groundbreaking study of Lewis et al. included 157 eyes of 157 OAG patients with a historical pressure of 21 mmHg or higher, with many of them on maximum tolerated medical therapy. Canaloplasty procedures were carried out on 121 eyes while 36 eyes underwent phacocanaloplasty. Of the 89 procedures performed with a successful placement of a suture, there was a 34% mean decrease in IOP from baseline (23.5 ± 4.5 to 15.5 ± 3.5 mmHg) and a 53% mean reduction in postoperative medications (1.9 ± 0.8 to 0.9 ± 0.9) at three-year follow-up. When phacoemulsification was combined with canaloplasty and successful suture placement, 27 eyes had a 42% mean decrease in IOP (23.5 ± 5.2 to 13.6 ± 3.6 mmHg) and an 80% mean reduction of postoperative medications (1.5 ± 1 to 0.3 ± 0.5).p < 0.0001). The outcomes in several recently published studies have equaled or surpassed those of the 2005 trial. In 2011, Grieshaber et al. published the results of a prospective study of 32 patients with OAG in which the mean IOP fell from 27.3 ± 5.6 mmHg preoperatively to 12.8 ± 1.5 mmHg at 12 months and 13.1 ± 1.2 mmHg at 18 months [Another long-term study by Bull et al. investigated the efficacy of canoloplasty and phacocanaloplasty on European eyes with open-angle glaucoma . One hun8 months . A more Compared to these previous trials, our large-scale study of 277 eyes with successful suture tensioning demonstrated a combined 23% reduction in IOP from baseline (19.7 ± 6.7 to 15.2 ± 4.3 mmHg) and 71% reduction in medication use (2.1 ± 1.2 to 0.6 ± 0.9) at three-year follow-up. The 150 patients undergoing canaloplasty alone had a 29% reduction in IOP (21.1 ± 7.2 to 15.0 ± 4.6 mmHg) and 77% reduction in medication use postoperatively (2.2 ± 1.3 to 0.5 ± 0.8) at three years. The 127 eyes undergoing phacocanaloplasty had a 17% mean decrease in IOP (18.1 ± 5.6 to 15.1 ± 4.0 mmHg) and a 65% reduction in medication use postoperatively (2.0 ± 1.1 to 0.7 ± 1.1) at three years. These patients had a lower baseline preoperative IOP as they were predominately undergoing surgical intervention for cataract, rather than increased pressure. In all cases, our study reached a similar endpoint when compared to previous studies, with a significant reduction in both IOP and postoperative medication use. This large data set, with 277 procedures performed by a single surgeon, acts to further verify and add to the efficacy of canaloplasty as a viable option for the treatment of OAG. A recent review by Cagini et al. summarizes the potential pitfalls associated with canaloplasty, noting that the meta-analysis of the literature demonstrates a relatively low rate of complications, particularly severe ones, compared to trabeculectomy . While tThis single-center clinical trial provides further evidence of the significant IOP lowering efficacy of canaloplasty with suture tensioning, with continued control through a 3-year postoperative period. The risk profile of canaloplasty was favorable and consistent with the well-documented, lower risks associated with other nonpenetrating procedures. Canaloplasty's safety profile and long-term efficacy make it a viable option for the majority of glaucoma patient types. It can be used in conjunction with existing drug-based glaucoma treatments, after laser or other types of incisional surgery, and does not preclude or affect the outcome of future surgery. It is a procedure that offers a high probability of success in effectively lowering IOP both alone and combined with phacoemulsification.


Background and Purpose: We recently noted a gradual change in the boundary electroencephalography (EEG) oscillation of 7.8 Hz between theta (θ) and alpha (α) bands in response to increased atherosclerosis levels in the elderly. The aim of this study was to investigate the role of boundary EEG oscillations of θ-α bands on cognitive functions in vascular cognitive impairments (VCI) patients.Materials and Methods: We examined 55 patients with VCI in carotid stenosis, and underwent EEG in a resting state with closed eyes for 5 min. The asymmetry index (AI) along homologous channel pairs  was assessed using neuronal activity topography (NAT). AI referring to 10 frequency components ranging from 4 to 20 Hz and neuropsychological assessments including linguistic competence were analyzed.Results: The main findings was that the language score had a positive association with AI in 7.8 Hz at F7-8 and a negative association with AI in 6.3 Hz at C3-4 and 14.1 Hz at F3-4.Conclusion: EEG asymmetry in a boundary range might have a special role in linguistic competence, suggesting the application of neural oscillation on the cognitive function evaluation and neurorehabilition induced by a frequency-specific transcranial alternating current stimulation. Electroencephalogram (EEG) signals are rich in information about electrical activity of neurons. In general, to signify the majority of EEG used in clinical practice, EEG oscillations are subdivided into the following bandwidths: delta (δ: 1–3 Hz), theta (θ: 4–7 Hz), alpha (α: 8–12 Hz), beta (β: 13–30 Hz), and gamma (γ: > 30 Hz) waves. Recently, we noted a gradual change in the boundary EEG oscillation of 7.8 Hz between θ and α bands in response to increased carotid atherosclerosis in community-dwelling elderly subjects  in carotid atherosclerosis is unclear and might be associated with diabetes. Previous EEG study in mild cognitive impairment (MCI) patients with diabetes found that resting-state functional connectivity is decreased in the alpha, and beta bands, and lower functional connection at intra-hemispheric distance mainly on the left hemisphere, suggesting a heterogeneous disruption of functional network structure , 72.6 ± 7.1 years]. Patients were selected based on the following criteria: (i) evidence of unilateral carotid stenosis of >60% (symptomatic or asymptomatic) confirmed with conventional angiography or computed tomography angiography and a degree of carotid artery stenosis determined using the criteria of the North American Symptomatic Carotid Endarterectomy Trial , visuospatial/constructional , language , attention , and delayed memory . Scores were normalized for age, gender, ethnicity, and education level, with a total score of 100 and an Fully informed consent in this study was obtained from all patients. The study design was approved by the ethics committee of Saiseikai Toyama Hospital . All subjects gave written informed consent in accordance with the Declaration of Helsinki. All methods were performed in accordance with the relevant guidelines and regulations.EEG recordings  were performed in an awake, resting state with eyes closed for 5 min, with 21 electrodes arranged according to the international 10–20 System . The contact impedance between the electrode and the scalp was <50 kΩ. The reference electrode was on the right earlobe, and then the average reference is computed as a mean of all electrodes. The raw EEG data were recorded at 0.08–300 Hz with 1,000 Hz sampling rate, and then the converted EEG data for NAT analysis were sampled at 200 Hz per channel and bandpass filtered to pick up signals in a frequency range of 4–20 Hz, which minimizes the effect of artifacts contaminating the recorded signals. The recorded signal sequence was divided into 0.64 s segments. To make the EEG data as high quality as possible and exclude the artifacts, each EEG finding was independently interpreted by EEG specialist who was blind to other data about the subjects except their age and sex. We carefully avoided particular epochs containing ocular movements, baseline shifts, drowsiness signs, and muscle or cardiac contamination.NAT provides rich information about neuronal activities by quantitative EEG analysis  was previously described in detail  such components NPSj, m defined asWe are going to derive dimensionless markers from the EEG signals which characterize stochastic nature of the neuronal activities generating these signals. The discrete power spectrum (PS) consists of the 10 frequency components NPSj, m >left and < NPSj, m >right were obtained from a left and right channel of a homologous channel pair with j = 1, 2, ···8, which evaluates absolute asymmetry along homologous channel pairs , and at frequency with m = 3,4, ···12, which evaluates the 10 frequency components ranging from 4.68 Hz through 18.72 Hz, respectively.Interhemispheric AI-value represents the balance between left and right brain activities which could be easily quantified using NPS. AI was measured between the following eight regions of interest: frontal , central (C3-4), temporal , parietal (P3-4), and occipital (O1-2) pairs. Furthermore, AI was banded into the following ten bands: theta , alpha , beta . Therefore, AI was calculated for each NPS using the following formula:®12 . Mean ± SD values of age, MMSE, RBANS were used as descriptive measures of normally distributed variables. Differences in mean total scores for AI among 3 groups composed of θ bands (4–8 Hz), α bands (8–13 Hz), β bands (13–20 Hz), and five groups composed of frontal lesion , central lesion (C3-4), temporal lesion , parietal lesion (P3-4), occipital lesion (O1-2) were compared by analysis of Tukey's test. Pearson's correlation and multiple linear regression analysis were used for statistical analyses. Firstly, Pearson's correlation was used to determine candidate predictors on the linguistic score. Variables with P < 0.05 in Pearson's correlation were included in the multiple linear regression analyses for predicting the language score. Secondly, the stepwise method based on F statistics was used for selection of variables for the final linear regression model. Entry and removal criteria were set at P < 0.05 and P > 0.10, respectively. Thirdly, ordinary least squares method is used in regression analysis to estimate a best fit model for predicting language scores from AI. Multicollinearity in the model was estimated using a Variance Inflation Factor (VIF), which of greater than five is generally considered evidence of multicollinearity. Differences with a P < 0.05 were considered statistically significant.All data were analyzed by JMPSD) on the MMSE and the RBANS in 55 VCI patients was 27.1 ± 1.5 and 74.5 ± 13.6, respectively. The mean scores of each index including immediate memory, delayed memory, attention, language, and visuospatial/constructional ability were 75.6 ± 15.2, 76.2 ± 16.7, 74.3 ± 14.2, 85.9 ± 6.9, and 87.8 ± 20.3, respectively. Because we were particularly interested in the relationship between EEG asymmetry and linguistic competence, Pearson's correlation was used between the language scores and AI at each channel pair in each frequency band for VCI patients.The mean score (±SD) of each AI at frontal , central (C3-4), temporal , parietal (P3-4), and occipital (O1-2) lesions in θ bands (4–8 Hz) were 0.024 ± 0.028, 0.028 ± 0.03, 0.037 ± 0.037, 0.035 ± 0.033, 0.027 ± 0.029, respectively. The mean scores of each AI including frontal , central (C3-4), temporal , parietal (P3-4), and occipital (O1-2) lesions in α bands (8–13 Hz) were 0.015 ± 0.023, 0.02 ± 0.031, 0.022 ± 0.025, 0.024 ± 0.029, 0.016 ± 0.02, respectively. The mean scores of each AI including frontal , central (C3-4), temporal , parietal (P3-4), and occipital (O1-2) lesions in β bands (13–20 Hz) were 0.007 ± 0.008, 0.008 ± 0.009, 0.011 ± 0.01, 0.009 ± 0.009, 0.007 ± 0.007, respectively.The mean scores (±P < 0.0001). The AI in temporal lesions  was significantly higher compared to that in frontal , central (C3-4), and occipital (O1-2) lesions, respectively (P < 0.0001).The AI in θ bands (4–8 Hz) was significantly higher compared to that inαbands (8–13 Hz) andβ bands (13–20 Hz), respectively (P < 0.05), respectively . studies have demonstrated that dominance language function of the left hemisphere may already be present in infants  and α (8–12 Hz) frequency ranges. However, the role of the boundary oscillation (7–8 Hz) between θ and α ranges still remains elusive and has not been adequately investigated. A previous study suggested that the boundary frequency of 6–8 Hz ranges is an anchor range that contributes the development of language ability in young children  is a promising neurophysiological marker of depression risk according to the level of trait anxiety  for several diseases, such as depression, ASD, stroke, and VCI. In clinical use of EEG asymmetry, the NAT system may provide supporting quantitative information about the higher degrees of complexity of parameter choice for tACS than that for transcranial direct current stimulation (tDCS). The present study suggests clinical application of tACS therapy for good recovery of cognitive functions, which could induce artificial EEG asymmetry via a specific oscillation of ~7.8 Hz in patients with cognitive impairments. Therefore, we wish to develop an integrated EEG (iEEG) system including neuromodulation and monitor the pre-post EEG changes. In the future, further improvements of the iEEG system for neurorehabilitation are needed to perform tACS, which induces artificial EEG using the most meaningful bands  for cognitive and motor recovery; and support an EEG monitor of physical and cognitive interventions to maintain and regain a healthy brain .The data that support the findings of this study are available from the corresponding author upon reasonable request. NAT is supported by Brain Functions Lab., Inc. (Mieko Tanaka, TS, TM, YuK, HM, and SK: study design; TS, MK, YH: data acquisition; TS, MT, HM, and YoK: analysis; TS and SK: interpretation of results. All authors contributed to revise and approve the final version of the manuscript and agree to be accountable for this work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Similarly, the CLint values of icaritin-3-O-glucuronide and icaritin-7-O-glucuronide in rat intestine microsomes (RIM) were 1.45 and 0.86 mL/min/mg, respectively. Taken altogether, dehydrogenation at isopentenyl group and glycosylation and glucuronidation at the aglycone were main biotransformation process in vivo. The general tendency was that icaritin was transformed to glucuronide conjugates to be excreted from rat organism. In conclusion, these results would improve our understanding of metabolic fate of icaritin in vivo. Icaritin is a naturally bioactive flavonoid with several significant effects. This study aimed to clarify the metabolite profiling, pharmacokinetics, and glucuronidation of icaritin in rats. An ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) assay was developed and validated for qualitative and quantitative analysis of icaritin. Glucuronidation rates were determined by incubating icaritin with uridine diphosphate glucuronic acid- (UDPGA-) supplemented microsomes. Kinetic parameters were derived by appropriate model fitting. A total of 30 metabolites were identified or tentatively characterized in rat biosamples based on retention times and characteristic fragmentations, following proposed metabolic pathway which was summarized. Additionally, the pharmacokinetics parameters were investigated after oral administration of icaritin. Moreover, icaritin glucuronidation in rat liver microsomes was efficient with CL Epimedium L. (Berberidaceae), are a widely used Chinese medicine for impotence, bone loss, and cardiovascular diseases  is the substrate concentration. Ksi is the substrate inhibition constant. The intrinsic clearance (CLint) was derived by Vmax/Km for Michaelis-Menten and substrate inhibition models.The parameters were as follows. + at m/z 391.1151 (C21H20O6Na) and [M+H]+ at m/z 369.1336 , the ion at m/z 313.0714 (C17H13O6) in positive ion mode was considered as the characteristic fragment ion .As had already been reported in the previous study , besidesOn the basis of MS/MS fragmentation pattern, the metabolites were deduced, clarifying the general metabolism in vivo. The extracted ion chromatograms (EICs) of prototype (M0) and metabolites (M1~M30) were shown in M0 (Parent Drug). M0  in biological samples was unambiguously identified by comparing with references. M15 and M23 (Hydration of Isopentene Group). Based on the [M+H]+ ion at m/z 387.1445  and [M−H]− ion at m/z 385.1286 (C21H21O7), the molecular formula of M15 (4.34 min) and M23 (4.97 min) was determined as C21H22O7, with one H2O more than M0. The MS/MS spectrum of [M+H]+ ion (C21H23O7) showed predominant [M+H−H2O]+ ion at m/z 369.1343 and 313.0715 , which indicated that M15 and M23 were the hydration products at isopentene group of icaritin and agreed with previous study of icariin [ icariin . M25 (Demethylation of Flavonoid Aglycone). According to the [M+H]+ ion at m/z 355.1180  and [M−H]− ion at m/z 353.1036 (C20H17O6), the formula of M25 (5.08 min) was supposed as C20H18O6, with a methyl group less than M0. The MS/MS experiments  showed a significant loss of neutral loss of C4H8 (56.0626 Da) from the ion at m/z 355.1180 to 299.0582 in positive ion mode or from the ion at m/z 353.1036 to 297.0373 in negative ion mode. Meanwhile, the demethylation position was purposed at 4′ position of B ring of flavonoid aglycone. Moreover, M25 was identified as desmethylicaritin by comparison of reference standard. M30 (Dehydrogenation of Isopentene Group). The formula of M30 (6.36 min) was C21H18O6, with two hydrogens fewer than M0, based on the [M+H]+ ion at m/z 367.1180  and [M−H]− ion at m/z 365.1047 (C21H17O6). In MS/MS spectrum , the ions at m/z 352.0948 and 313.0721 were attributed to obvious loss of CH3 (15.0235 Da) and C4H6 (54.0470 Da) group, respectively, which indicated that the dehydrogenation position was at isopentene group [M27~M29 (Hydroxylation of Isopentene Group). From the [M+H]+ ion at m/z 385.1288  and [M−H]− ion at m/z 383.1175 (C21H19O7), the formulae of M27 , M28 (5.54 min), and M29 (5.66 min) were speculated as C21H20O7, which was one oxygen more than M0. In (+) ESI-MS/MS spectrum , the ion at m/z 385.1288 could lose a H2O and C4H6 group to produce the daughter ions at 367.1186 ([M+H−H2O]+) and 313.0715 ([M+H−C4H6]+), respectively. This illustrated that M27~M29 were tentatively characterized as the hydroxylated products of M0 at the isopentene group. M14, M16, M17, M19, M20, and M21 (Glycosylation of Flavonoid Aglycone). M21 (4.88 min) was given a [M+H]+ ion at m/z 515.1927  and [M−H]− ion at m/z 513.1766 (C27H29O10) in full scan mass spectrum. The ion at m/z 515.1927 could easily yield the characteristic fragment ions at m/z 369.1340 and 313.0726 by subsequent loss of C6H10O4 and C4H8 . So M21 could be the glycosylation product of M0 by adduct of rhamnose . Similarly, M17  was the glucose conjugate of M0, while M19  and M20  were the xylose and rhamnose glycosylation derivates of M21, respectively. M14, M17, and M21 were identified as epimedin C, icariside I, and icariside II, respectively.λmax 270 nm, C39H51O19, −0.1 ppm) and M16  both with the formula of C39H50O19  were tentatively characterized as the glucose glycosylation conjugate of M20. These glycosylation reactions were the same as the metabolism of epimedin C in rats reported in reference . By comparing with references, M14, M17, and M21 were identified as epimedin C, icariside I, and icariside II, respectively. And the MS/MS spectra of M17, M19, and M20 were shown in Figures S1h–S1j, respectively.M14 . In full scan mass spectrum, M13 (4.34 min), M18 (4.57 min), and M26 (5.10 min) all exhibited the [M+H]+ ion at m/z 545.1663  and [M−H]− ion at m/z 543.1501 (C27H27O12) with a formula of C27H28O12 of 176.0325 Da larger than M0. The MS/MS spectrum  displayed an obvious loss of C6H8O6 group from parent ion at m/z 545.1663 to the daughter ion at m/z 369.1338, which suggested an existing glucuronic acid of these three metabolites. Just like reported studies [33H36O18, −0.3 ppm) and M8   were characterized as diglucuronidation derivates based on two molecules of C6H8O6 fragment larger than M0.λmax 269 nm, C27H30O13, 0.5 ppm) and M10  with the MS/MS spectrum shown in Figure S1 m were tentatively considered as the monoglucuronidation products of M15 and M23. M3  and M6  were characterized as monoglucuronide conjugate of M25. The MS/MS spectrum of M3 and M6 was exhibited in Figure S1n. Meanwhile, M4 , M5 , M7 , M9 , and M11  were regarded as monoglucuronidation derivates of M27~M29 and their MS/MS spectrum was displayed in Figure S1o. M12 , M22 , and M24  were tentatively identified as glucuronidation conjugates of M30. And their MS/MS spectrum was shown in Figure S1p.Similarly, M2  versus respective plasma concentrations (x) using a 1/x2 weighting factor and linear least-squares regression analysis. A series of standard solutions were used to generate calibration curve. The correlation coefficients (r2) of calibration curves were greater than 0.9926 within 2.0~512.0 ng/mL and LLOQ was 2.0 ng/mL. The regression equations, correlation coefficients, and LLOQ were shown in Table S1.The LOD and LOQ were calculated as 3-fold and 10-fold of the ratio of signal-to-noise, respectively. The LLOQ was defined as the lowest concentration in the calibration curve with accuracy of 80~120% and precision of 20%. Calibration curves were acquired by plotting peak area  indicated that icaritin were stable under different storage conditions.Cmax was (294.5 ± 22.7) ng/mL when Tmax was (5.3 ± 1.1) h after oral administration. The area under the concentration-time curve (AUC∞0−) and mean residence time (MRT0-∞) were (3145.0 ± 302.3) ng·h/mL and (10.9 ± 1.3) h, respectively. The results illustrated that icaritin had a poor absorption after oral administration. The reason may be that icaritin stepped into small intestine to undergo mass phase I and phase II metabolism by intestinal flora, especially the glycosylation and glucuronidation conjugates.The mean concentration-time profiles of these bioactive components were shown in μM, with LOD (S/N = 3~5) and LOQ (S/N = 8~10) of 0.01 and 0.02 μM, respectively. And the acceptable linear correlation (Y = 12149X) was confirmed by correlation coefficients (r2) of 0.9994. The accuracy and precision of the intraday and interday error were both less than 3.4%. There were no matrix effects observed and no other sample preparation performed except those mentioned in the manuscript.Due to lack of reference standard, quantification of icaritin glucuronide was based on the standard curve of the parent compound (icaritin) according to the assumption that parent compound and its glucuronide have closely similar UV absorbance maxima –29. The O-glucuronide (M13) and icaritin-7-O-glucuronide (M18) in RLM was well modeled by the substrate inhibition equation , following the substrate inhibition kinetics with Km values of 3.62 and 1.53 μM, respectively. Similarly, the CLint values of M13 and M18 in RIM were 1.446 and 0.861 mL/min/mg, respectively, whereas the Km values of M13 and M18 in RIM in Michaelis-Menten model were 8.22 and 9.56 μM, respectively. In addition, Ki values of M13 and M18 in RLM were 11.31 and 17.07 μM, respectively. The detailed parameters of M13 and M18 were listed in Kinetic profiling revealed that formation of icaritin-3-equation , whereasequation . In cont M0, M1, and M13 were the main xenobiotics in plasma  as an indicator of enzymes activity was advantageous, because (1) CLint represents the catalytic efficiency of the enzyme and is independent of the substrate concentration; (2) compared with other kinetic parameters such as Km and Vmax, CLint is more relevant in an attempt to predict hepatic clearance in vivo [int values were used to determine icaritin glucuronidation activity in this study.Characterization of icaritin glucuronidation assumed a great role in the understanding of its pharmacokinetics and bioavailability. Oral bioavailability is a major factor in determining the biological actions of icaritin in vivo following oral administration of the compound . This stt values . Use of  in vivo . TherefoBased on the metabolite profiles, the metabolic pathways of icaritin were proposed and shown in int values of 1.12 and 1.56 mL/min/mg for M13 and M18, respectively. Similarly, the CLint values of M13 and M18 in RIM were 1.45 and 0.86 mL/min/mg, respectively. Taken altogether, this study could provide an experimental basis to understand the metabolic fate of icaritin in rat.As a result, a total of 30 metabolites were identified or tentatively characterized based on the retention time behaviors and fragmentation patterns. Dehydrogenation at isopentenyl group and glycosylation and glucuronidation at the flavonoid aglycone were the main biotransformation process of icaritin in vivo. Meanwhile, a validated method was successfully applied to a pharmacokinetic study. Moreover, icaritin glucuronidation in RLM was efficient with CLn = 6). Table S3: Intra- and inter-day precision and accuracy test of icaritin in rat plasma. Table S4: Stability test of icaritin in rat plasma under different condition (n = 3). Figure S1: (+) ESI-MS and MS/MS spectra of M0~M30. Figure S2: EICs of M0~M30 in rat intestine samples. Figure S3: Specificity test of icaritin in rat plasma.Table S1: Leaner range and LLOQ test of icaritin in rat plasma. Table S2: Matrix effect and recovery test of icaritin in rat plasma (


The optimal doping concentration of F in Na3V2(PO4)3 is 0.15 mol %. By neutron powder diffraction data, the chemical composition of as-synthesized material is Na2.85V2(PO3.95F0.05)3. The half-cell of Na2.85V2(PO3.95F0.05)3 cathode exhibits a stable discharge capacity of 103 mAh g−1 and 93% of capacity retention over 250 cycles without decay at 0.1 A g−1, which is higher than that of bare Na3V2(PO4)3 (98 mAh g−1). The high rate capability of Na2.85V2(PO3.95F0.05)3 is also dramatically enhanced via increase the conductivity of host material by F-doping. Moreover, the symmetrical Na-ion full-cell is fabricated using Na2.85V2(PO3.95F0.05)3 as cathode and anode materials. It is achieved that the good reversibility and superior cycling stability about 98% of capacity retention with ~100% of coulombic efficiency at 1.0 A g−1 throughout 1000 cycles. These results demonstrate that the optimal amount of Na2.85V2(PO3.95F0.05)3 is a distinctive potential candidate for excellent long-term cyclic stability with high rate low-cost energy storage applications.A series of Na Among those batteries, sodium-ion batteries (SIBs) have been attracted significant interest as a promising alternative to lithium-ion batteries (LIBs) for merits of natural abundance, environmental benignity and similar intercalation chemistry with lithium3. Polyanion phosphate-based cathode materials, such as NaVOPO4F0.5, Na2FePO4F, Na3V2(PO4)2F and Na3V2(PO4)2F3, recently, have been received much attention due to their excellent structural, good thermal stabilities and strong networks8. A NASICON-type Na3V2(PO4)3 is one of the most potential cathode material for Na-ion storage. Na3V2(PO4)3 exhibits two distinct potential plateaus in the V4+/V3+ and V3+/V2+ redox couples at 3.4 V and 1.6 V vs. Na/Na+, corresponding to theoretical capacities of 117.6 and 58.8 mAh g−1, respectively9. Moreover, the pristine Na3V2(PO4)3 suffers from severe capacity fading at high rate because of the low electrical conductivity and poor ion diffusivity10. To improve rate capability and cycle life of Na3V2(PO4)3, much efforts have been made by different approaches, such as doping of other cation or anion, including K+, Al3+, Fe3+, Mn2+, Mg2+ or B3+17, carbon-coating using various carbon precursors and reducing the particle size via optimizing synthetic processes23. The substitution of other metal/non-metal in Na3V2(PO4)3 are, indeed, quite efficient way to enhance electrochemical properties compared to bare one. Additionally, some of cation substitutions are unstable or toxic, need to be strongly electronegative enough to reduce the valence of vanadium ion and electrochemically active at similar potential not to sacrifice the gravimetric capacity. Therefore, the fluorination replacing O2− ions by F− ions was a rational alternative. The fluorine-based polyanion frameworks could be enhanced the working potentials and larger iconicity of transient metal-F− bond compared to metal-oxide17. It is noteworthy that the modification of polyanion groups by F− and their electrochemical affection factors studies are still quite interesting of identification and development of battery materials.Over the past decades onwards, Li-ion batteries have been widely used for various energy sectors such as portable electronics and transport applications. Conversely, the limited stock of lithium source availability and increasing cost may restrict their large-scale electrical energy storage systems. Numerous researchers have been focused on alternative batteries, such as Na-ion, K-ion, Al-ion, Mg-ion batteries and so onx3-V2(POx4-Fx)3  polyanion cathode materials were synthesized via a simple sol-gel method and evaluate the electrochemical performance for sodium-ion storage. Interestingly, the optimal fluorine in NVP material is improved the conductivity and induce the catalytic activity of pristine NVP. In addition, the symmetric full-cell was constructed via optimal amount of F-doped NVP used for cathode and anode material. It delivers the outstanding ultra-long term cycle performance.In this study, Na4VO3 was dissolved in distilled water under constant stirrer at 80 °C and yellow solution was observed. Then, citric acid was added drop by drop into the (VO3)− aqueous solution and the color of solution was gradually changed from yellow to dark blue24. Afterwards, other precursors of Na, PO4 and F (for doping) were added into the solution. The mixed solution was constantly stirrer at 80 °C and formed as a homogeneous solution to viscous gel. Subsequently, as-prepared gel was dried in oven. Finally, sample was preheated at 350 °C for 4 h and calcined at 800 °C for 8 h in Ar. At the end, we could obtain a series of Nax3-V2(POx4-Fx)3 powder samples.Figure 3V2(PO4)3 materials and the corresponding observed structural parameters are shown in Table R-3c27. The refined unit cell lattice parameters are presented in Fig. a’ axis (a = b axis) and the lattice constants of anionic (F−) atom doped NVP samples were slightly increased than pristine NVP. It may be related to the small amount of F-substitution with relatively smaller ionic radius (F− = 1.33 Å) for O2− (r = 1.40 Å) anion and larger than V3+ (0.64 Å)/V2+ (0.79 Å), Na+(1.02 Å) cations, respectively. Furthermore, the confirmation of F-doped sites was studied by Neutron powder diffraction analysis and the result of pristine and 0.15% F-doped NVP are shown in Fig. Figure 3V2(PO4)3 and Na2.85V2(PO3.95F0.05)3, respectively  method. The surface area of NVP and F-doped NVP were measured to be 8.3 m2 g−1 and 8.8 m2 g−1, respectively. The average pore size was found to be 25.64 and 15.68 nm of bare and NVP-F0.15 sample using Barrett-Joyner-Halenda (BJH) method. The results show that both specific surface area and pore size distribution of NVP w/wo doping were similar33.The pore size distribution and specific surface area were characterized using N3V2(PO4)3 and Na2.9V2(PO3.967F0.033)3, Na2.85V2(PO3.95F0.05)3, Na2.7V2(PO3.9F0.10)3 were determined to be 1.63 × 10−6, 2.95 × 10−6, 6.33 × 10−5 and 1.67 × 10−−6 S/cm, respectively. Among these samples, the Na2.85V2(PO3.95F0.05)3 demonstrated better electrical conductivity. Fig. 0.15 electrodes at a scan rate of 0.1 mV s−1. The oxidation (Na extraction) and reduction (Na insertion) peaks were located at 3.45 V and 3.30 V for NVP and NVP-F0.15 electrode materials, respectively. The average voltage (ΔE) was observed at 3.37 V for both electrodes. It is close to the equilibrium voltage of V4+/V3+ redox couple in NVP26. It designates that the deintercalation/intercalation of two sodium-ions located at the M2 sites into a single formula unit of the prepared electrodes26. The potential difference (ΔV) was 0.15 V for NVP and 0.16 V for NVP-F0.15 electrodes. CV curves illustrate noticeably anodic peaks move to a lower potential, while the cathodic peaks slightly shift to higher potential, as a result indicates the inductive effect characteristic of NVP35. The diffusion coefficient of sodium-ion in NVP could be calculated from the linear relationship between their peak currents (ip) and the square root of scan rates (v1/2) using the following Randles-Sevcik equation via CV analyses:37.ip is the peak current (A), m is the mass of the active cathode material, F is Faraday constant, R is gas constant, T is absolute temperature, n is number of electrons involved in the reaction (n = 2), A is the effective contact area between the electrode and the electrolyte, as obtained from the cathode material multiplied with the active mass ratio, and C is the concentration of Na ions in the cathode, as calculated from the crystallographic cell parameters of NVP. CV curves of bare NVP and NVP-F0.15 electrodes with different scan rate are shown in Fig. v1/2) and peak current (ip) are shown in Figure 0.15 samples were 5.68 × 10−10 cm2 s−1 and 6.37 × 10−10 cm2 s−1 for anodic and 6.03 × 10−10 cm2 s−1 and 6.46 × 10−10 cm2 s−1 for cathodic reaction, respectively. These results are in consistent with NVP reports37.The conductivity of active material is very important to Na-ion intercalation/de-intercalation processes. The electronic conductivity of as-synthesized samples was measured by using four-probes DC method in room temperature. The corresponding result is shown in Fig. 0.15 electrodes. The Nyquist plot with corresponding fitted equivalent circuit model is presented in Fig. s is the electrolyte resistance and CPE represents the double layer capacitance and capacity of the surface layer. Rct is the charge transfer resistance. The Warburg impedance (W) represents the diffusion behaviour at low frequencies32. The semicircle portion of the impedance plot defines the polarization of the electrode-electrolyte interface, which became smaller after F-doped into NVP. The charge transfer resistance of NVP-F0.15 (52 Ω) electrode was lower than that of bare NVP (276 Ω). Thus, by using F-doping in NVP material, charge transfer and ion transport kinetics could be effectively improved. In addition, An Exchange current density (i0) is one of the main factors to affect the kinetic process, which can be used to measure the catalytic activity of electrodes. The following equation describes the calculation of Exchange current density (i0)38:0) values were 0.0465 and 0.2470 mAh g−1 for bare and NVP-F0.15 samples, respectively. This result represents the F-doping to induce the catalytic activity of bare NVP.EIS was further used to investigate the electrochemical kinetics behaviour of fresh bare NVP and NVP-F0.15 electrodes at 1st, 25th,50th, 100th, 200th and 250th cycles in the current of 0.1 A g−1. The voltage curve has a flat charge plateau at about 3.4 V (0.1 A g−1), which corresponds to the redox of V4+/V3+ couple. This result is good agreement of CV results39. The initial discharge capacity of NVP and NVP-F0.15 electrodes were 98 mAh/g and 103 mAh/g, respectively  and higher (NVP-F0.30) content of fluorine doped NVP electrodes exhibited capacity of 85 and 82 mAh/g, as shown in Fig. 0.10, NVP-F0.15 and NVP-F0.30, respectively  The activation process for the cell component during the electrochemical reactions to fluctuates the coulombic efficiency;43 (iii) The electrolyte decomposition and side reactions of PC based electrolyte systems44 and (iv) The dissolution of oxidized NVP for that the reaction of NVP can be fully ensued before the occurrence of oxygen evolution45. Moreover, the optimization of the suitable electrolyte is an effective vital role of coulombic efficiency improvement in SIBs46. The coulombic efficiency was observed 99% after initial few cycles and it was maintained upto 250 cycles of bare NVP and F-doped NVP electrodes . After these C-rate tests, the discharge capacity of NVP-F0.15 sample maintained around 102 mAh g−1 at 0.1 A g−1 over the 30 cycles. The capacity retention was observed 98% with more stable cyclic performance at higher C rate of 1 A g−1.Figure ely Fig. . Howeverely Fig. . The corely Fig. . The coudes Fig. . The F-ddes Fig. . It is e0.15 as cathode and anode materials was carried out in our study. Figure −1. The oxidation peaks were observed 1.72 V and 1.78 V and the reduction peaks were 1.68 and 1.60 V, which were good agreement with the output voltage plateau of charge/discharge curve (V3+/V2+ for redox reactions 1.6 V) and earlier reports51. Fig. −1. The charge/discharge capacity was 74/60 mAh g−1 with retention about 98% over the 1000 cycles at 1.0 A g−1, respectively. The first three charge/discharge profiles revealed the voltage plateaus of charge/discharge were appeared at ~1.78 V and ~1.69 V, which were very close to the CV values and previously reports50. As shown in Fig. −1 over the 2500 cycles and inset of right hand side in Fig. 0.15//NVP-F0.15 symmetric full-cell.To approach practical application, symmetric full-cell fabricated by using NVP-F0.15 electrode for both half and full-cells could be ascribed to (i) the higher electronic conductivity, (ii) improves the kinetics and catalytic behaviour of bare NVP, (iii) the slight lattice alteration of NASICON structure permits a fast transport of Na ions. Besides, the transition metal of V provides a wide range of charge compensation during the Na+ intercalation process. These are favourable to enhance the ionic conductivity. The long-terms cycling stability owing to the protective surface films on the electrode materials were formed in the PC based electrolyte system, which was beneficial for the improved passivation and suppression of side reactions between the Na metal and the PC based electrolyte solution containing Na salts40.The excellent electrochemical performance of NVP-F0.15) sample was observed porous morphology, superior conductivity and better catalytic effect than bare NVP. In half-cell Na-ion storage, the NVP-F0.15 electrode exhibited higher capacity, good cycle life and high rate capability than rest materials. It was observed initial discharge capacity of 103 mAh g−1 and bare NVP electrode was 98 mAh g−1 at 0.1 A/g. The symmetric Na-ion full-cell was fabricated using NVP-F0.15 as both cathode and anode material. The charge/discharge capacity was observed 74/60 mAh/g with retention about 98% over the 1000 cycles. Afterwards, the retention was reduced to 83% throughout the 2500 cycles and 100% of coulombic efficiency was maintained at 1.0 A/g, respectively. Overall this investigation, the superior appealing features of optimal F-doped NVP dual electrode material, such as high rate capability and long-term cyclic stability, will qualify their utilization in the advanced large-scale low cost energy storage applications.A series of NASICON-type F-doped sodium vanadium phosphate polyanion materials were successfully synthesized by simple sol-gel method and the results were compared with bare material. The rhombohedral crystalline phase of both samples was observed by X-ray diffraction techniques. The lattice parameters of F-doped samples were slightly changed, which represented the small amount of substitution on the host material. The neutron powder diffraction results demonstrated that the oxygen at two 36f sites was partially substituted by F-ions with 5% of sodium content deficiency in the F-doped sample. The presences of fluorine and vanadium state were confirmed by XPS. The optimal F-doped NVP , ammonium vanadate , ammonium dihydrogen phosphate , sodium fluoride  and citric acid  were used as precursor materials. Sol-gel method was used to synthesize a sequence of Nax3-V2(POx4-Fx)3, where x = 0, 0.1, 0.15 and 0.3 3 materials, NH4VO3 was dissolved first into distilled water and then the solution was stirred at 80 °C. After a clear solution formed, appropriate amount of citric acid, NH4H2PO4, Na2CO3 and NaF solutions were drop by drop added into the NH4VO3 solution one by one with vigorously stirring at 80 °C. The gel was formed after several hours. The obtained gel was ground in a mortar and preheated at 350 °C for 4 h. Then, the powder was again ground and calcined at 800 °C for 8 h in flowing argon atmosphere. Finally, we can obtain F-doped Na3V2(PO4)3 powder. For comparison, bare NVP sample (No NaF adding) was synthesized by the same processes. Nax3-V2(POx4-Fx)3  are represented as bare NVP, NVP-F0.10, NVP -F0.15 and NVP -F0.30, respectively.Sodium carbonate (Nax3-V2(POx4-Fx)3 samples were studied by powder X-ray powder diffraction (XRD) using a Bruker D8 diffractometer with monochromatic Cu Kα radiation (λ = 1.54060 Å) operated at 40 KV and 30 mA. The powder XRD data were collected in a 2θ range from 10° to 80°. Neutron powder diffraction (NPD) data were collected on the high resolution powder diffractometer Echidna at the OPAL facility  using neutrons of wavelength 2.4395 Å selected from the Ge (331) monochromator52. The two powder samples were loaded into the standard 9-mm vanadium cans, and data were collected at room temperature. A vacuum chamber was used for reducing the air scattering. Both XRD and neutron diffraction patterns were analyzed using the General Structure Analysis System (GSAS) program, following the Rietveld refinement method. The morphological observation of prepared materials were characterized via field emission scanning electron microscopy  with energy dispersive spectrometer/electron mapping  and high-resolution transmission electron microscopic (HR-TEM)  technique. Chemical valence states of the elements were investigated by X-ray photoelectron spectroscopy . A Tristar 3000 accelerated surface area and porosimetry instrument was used to measure the prepared products N2 adsorption/desorption isotherms.The crystal structure and phase purity of a series of Na4: PC | NVP (or NVP-F) and symmetric full-cell of NVP-F0.15| NaClO4: PC | NVP-F0.15 were used to evaluate the electrochemical performance via CR2032 type coin cells. The cathodes (positive electrodes) were prepared by mixing of 70 wt. % of active material (NVP or NVP-F powder), 10 wt. % of Super P and 20 wt. % of Poly-(vinylidene fluoride)  in N-methylpyrrolidone  solvent to form a homogeneous slurry. Afterwards, the mixed slurry was spread uniformly on a thin aluminum foil and dried in vacuum at 120 °C for 6 h and then roll pressed. The samples were punched into circular discs. Glass fiber  as separator was drenched in the electrolyte for 24 h prior to use. The coin cell assembling procedures were performed using Ar-filled glove box by keeping both the oxygen and moisture levels less than 1 ppm. The galvanostatic charge-discharge measurements were performed using a AcuTech battery testing system  in the potential range of 2.8–3.8 V (V vs. Na+/Na) at ambient temperature with different C-rates. The cyclic voltammograms (CV) were measured by CH Instruments Analyzer CHI 6273E at a scan rate of 0.1 mVs−1 voltage window between 2.8 V and 3.8 V. The AC impedance was tested in the range from 0.1 Hz to 100 KHz with the amplitude of 5 mV. The symmetric Na-ion full-cell was fabricated using NVP-F0.15 as both electrode materials and evaluated the cycling performance. The cathode/anode weight ratio was maintained 1:2. The current density (A g−1) and capacity values (mAh g−1) presented throughout this study were calculated based on the total mass of the electrode materials.A half-cell assembly of Na-metal | NaClOSupporting data


Loktanella cinnabarina strain XM1, which was isolated from coastal surface water and shared 99.43% 16S rRNA gene sequence identity with the deep-sea bacterium L. cinnabarina LL-001T. The estimated genome size of strain XM1 is 3,782,785 bp, with a G+C content of 67.9%.We report here the draft genome sequence of  Loktanella, a member of the Rhodobacter group within the class Alphaproteobacteria, was first described by Van Trappen et al. in 2004 , and has 99.43% 16S rRNA gene sequence identity with the deep-sea bacterium L. cinnabarina LL-001T. Colonies of L. cinnabarina strain XM1 are pink in color, while those of L. cinnabarina LL-001T are vermilion.The genus  in 2004 . To dateeawater 24, sea saeawater 2, 6, sedieawater 2, marine eawater 2, and miceawater 2. In partsediment . Here, wde novo using Newbler assembler version 2.8. GapCloser software was used to close gaps between the contigs and to generate final assembled sequences, which were then automatically annotated using the Rapid Annotations using Subsystems Technology (RAST) version 2.0 server . The draft genome sequencing of strain XM1 was performed at the Personal Biotechnology Co., Ltd.  with an Illumina MiSeq platform using a 2 × 301-bp paired-end DNA library. Sequencing resulted in a total of 3,039,150 reads and 752,681,545 nucleotide bases. Filtered reads were then assembled 0 server .N50 contig length is 134,038 bp. Total genome length and average G+C content (67.9%) are consistent with those of other sequenced Loktanella isolates  value is observed between strain XM1 and L. hongkongensis DSM 17492T (86.88%) than between strain XM1 and L. cinnabarina strain LL-001T (86.12%), as determined by the online ANI calculator (L. hongkongensis DSM 17492T, isolated from a marine biofilm in Hong Kong waters (Loktanella species isolated from different environments, we believe that further analysis will shed new light on the lifestyle and evolution of this widely distributed genus.The draft genome contains 92 contigs with a total length of 3,782,785 bp and a coverage depth of 89×. The largest contig is 585,710 bp, and the isolates . The cod-001T 86.%, as detNQWH00000000. The version described in this paper is the first version, NQWH01000000.This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under accession number