There is considerable evidence that many people take dietary supplements including those of herbal origin as an alternative therapy to improve their health. One such supplement, with an amalgam of constituents, is CellGevity®. However, the effect of this dietary supplement on drug-metabolizing enzymes is poorly understood, as it has not been studied extensively. Therefore, we investigated the effect of CellGevity dietary supplement on selected rat liver microsomal cytochrome P450 (CYP) enzymes, the most common drug-metabolizing enzymes. We also determined the total antioxidant potential of this dietary supplement To determine the antioxidant potential of CellGevity dietary supplement, 2,2-diphenyl-2-picryl-hydrazyl (DPPH), total phenolic, and flavonoid assays were used after initial preparation of a solution form of the supplement . Rats received oral administration of these doses of the supplement for 7 days, after which the effect of the supplement on selected liver CYP enzymes was assessed using probe substrates and spectroscopic and high-performance liquid chromatographic methods. Rats which received daily administration of 80 mg/kg of phenobarbitone and distilled water served as positive and negative controls, respectively.50 value of the supplement 0.34 ± 0.07 mg/ml compared to 0.076 ± 0.03 mg/ml of the BHT (positive control). The total phenolic content of the supplement at a concentration of 2.5 mg/ml was 34.97 g gallic acid equivalent (GAE)/100 g while its total flavonoid content at a concentration of 2.5 mg/ml was 6 g quercetin equivalent (QE)/100 g. The supplement significantly inhibited rat CYP2B1/2B2 , CYP3A4 , and CYP2C9  while it had no significant inhibitory effect on CYPs 1A1/1A2, CYP1A2, and CYP2D6. The IC in vitro and has an inhibitory effect on selected rat liver CYP enzymes, suggesting its potential interaction with drugs metabolized by CYP enzymes. CellGevity dietary supplement possesses moderate antioxidant activity Noncommunicable diseases (NCDs) such as cardiomyopathies, asthma, diabetes mellitus, and cancer are the most common causes of death globally, with a higher percentage of premature deaths happening in developing nations than in developed nations . This hiThe use of alternative therapies, in the form of dietary supplements, is becoming very common throughout the world, as many people nowadays are adopting a variety of lifestyle habits that contribute to healthy living . Dietaryβ-carotene and vitamin E . The HPLC conditions for the assay comprised an injection volume of 50 μL, a flow rate of 0.8 ml/min, a C18 column , a temperature of 40°C, and a maximum pressure of 40 bar. A diode array served as the detector. Products formed were measured using an isocratic HPLC method. The mobile phase consisted of 60% of 20 mM potassium phosphate buffer (pH 7.4), 22.5% methanol, and 17.5% acetonitrile.g et al. . Brieflyp values < 0.05 were considered statistically significant. Significant differences were calculated with Bonferroni's Multiple Comparison Tests, and graphs were produced using Graph Pad Prism Software Version 7 .All values are expressed as mean ± standard deviation (SD). Differences between groups were tested for significance using a One-Way ANOVA. 50) was 0.34 ± 0.07 mg/ml compared to 0.076 ± 0.03 mg/ml of the BHT (positive control). The total phenolic content of the supplement at a concentration of 2.5 mg/ml was 34.97 g gallic acid equivalent (GAE)/100 g while its total flavonoid content at a concentration of 2.5 mg/ml was 6 g quercetin equivalent (QE)/100 g. Figures 50 values of CellGevity as compared to BHT.To evaluate the antioxidant potential of CellGevity dietary supplement, DPPH, total phenolic, and flavonoid assays were used. The concentration of the supplement required to inhibit 50% of free radicals  enzyme activities, selected CYP assays were used. CYP1A1/1A2 and CYP1A2 Assays. There was no significant difference in the activity of CYP1A1/1A2 enzyme among NC, LDT, and HDT groups (p > 0.05). However, the CYP1A1/1A2 enzyme activity of these three groups markedly decreased compared to PC group (p < 0.05). A similar observation was made in CYP1A2 enzyme activity . A similar pattern was observed in p < 0.05), that in CYP3A4 in p > 0.05). CYP2D6 and CYP2C9 Assays. CYP2D6 enzyme activity in LDT group increased significantly compared to HDT group (p < 0.05). In addition, NC group showed a markedly high CYP2D6 activity in comparison with PC group (p < 0.001). As seen in p < 0.05). Overall Effect of the Supplement on Rat CYP Enzymes. The overall effect of this supplement on selected CYP enzymes is summarized in In the current study, we evaluated the antioxidant potential of CellGevity dietary supplement, comprising an aggregate of ingredients, and the effect of this supplement on the activities of selected rat liver microsomal enzymes. This study focuses on CYP enzymes (one of the conserved entities among species) which are the main enzymes involved in numerous oxidative reactions and often play a critical role in the metabolism and pharmacokinetics of xenobiotics. It is well established that some rat CYP enzymes are closely related to those of humans. For example, CYP1A shows a strong conservation among species with an identity to human > 80% in rat  , 28.Some constituents of CellGevity dietary supplement, such as curcumin, resveratrol, milk thistle, quercetin, and piperine, are known inhibitors of CYP3A4 , 29–31. Our study also showed that CellGevity dietary supplement significantly inhibited rat CYP2B1/2B2. Curcumin, one of the constituents of the supplement, is a less potent inhibitor of rat CYP2B1/2B2 compared to CYP1A1/1A2 enzyme . This eaAnother CYP enzyme, significantly inhibited by CellGevity dietary supplement, was CYP2C9. Although the effect of individual constituents of this dietary supplement on CYP enzyme activity was not investigated in our study, rodent and human microsome studies have shown that resveratrol, a constituent of this dietary supplement, is a potent inhibitor of CYP2C9 , 38. A d50 value of CellGevity dietary supplement was 0.34 ± 0.07 mg/ml compared to 0.076 ± 0.03 mg/ml of the BHT. The total phenolic content of the supplement at a concentration of 2.5 mg/ml was 34.97 g gallic acid equivalent (GAE)/100 g while its total flavonoid content at a concentration of 2.5 mg/ml was 6 g quercetin equivalent (QE)/100 g. This antioxidant potential is moderately high compared to a related study where the authors reported a synergistic antioxidant activity of a green tea of herbal origin determined by an EC50 value of 33.5 mg/ml, a total phenolic, and flavonoid content of 2.5 g GAE/10 g and 1.2 g QE/10 g, respectively [ in vitro assays [ in vivo. Once in circulation, riboceine is metabolized into cysteine and ribose, which are transported into cells [In the current study, the ECectively . Furthero assays . Howeverto cells . It is nto cells , suggest in vitro and also inhibits activities of rat liver CYP2B1, CYP3A4, and CYP2C9 enzymes. Inhibition of these selected CYP enzymes by this dietary supplement suggests the possibility that CellGevity dietary supplement may contribute to supplement (herb-) drug interactions in humans.In conclusion, this study reports that CellGevity dietary supplement possesses antioxidant property


Neisseria meningitidis (MenB), the predominant cause of meningococcal disease in wealthy countries. MenB ChAs exploit factor H binding protein (fHbp) as a molecular scaffold to display the immunogenic VR2 epitope from the integral membrane protein PorA. Structural analyses demonstrate fHbp is correctly folded and the PorA VR2 epitope adopts an immunogenic conformation. In mice, immunisation with ChAs generates fHbp and PorA antibodies that recognise the antigens expressed by clinical MenB isolates; these antibody responses correlate with protection against meningococcal disease. Application of ChAs is therefore a potentially powerful approach to develop multivalent subunit vaccines, which can be tailored to circumvent pathogen diversity.There is an urgent need to develop vaccines against pathogenic bacteria. However, this is often hindered by antigenic diversity and difficulties encountered manufacturing membrane proteins. Here we show how to use structure-based design to develop chimeric antigens (ChAs) for subunit vaccines. ChAs are generated against serogroup B  N. meningitidis vaccines. Here the authors graft the VR2 loop of PorA onto an fHBp-based scaffold to demonstrate proof-of-principle of a chimeric antigen strategy and vaccination against meningococcal disease.Factor H binding protein (fHbp) and PorA are components of experimental serogroup B  However, challenges remain in developing vaccines against pathogens for which toxoid and capsule-based vaccines are not feasible. These pathogens include non-typeable strains of H. influenzae and S. pneumoniae3, un-encapsulated pathogens such as Neisseria gonorrhoeae and Moraxella catarrhalis6 and encapsulated serogroup B N. meningitidis, for which a capsule-based vaccine is not feasible7. Given the rise in the emergence of multi-drug resistant bacteria8, new approaches for vaccine development are required. However, strategies for generating successful vaccines are hampered by pathogen diversity9 and the difficulties associated with presenting epitopes from membrane-embedded surface proteins to the immune system10.Toxoid and capsule-based vaccines have prevented millions of deaths caused by bacterial pathogens. Toxoid-based vaccines have almost eliminated diphtheria and tetanus in wealthy countriesN. meningitidis; outer membrane vesicle vaccines (OMVV) and recombinant protein subunit vaccines. OMVVs were first developed in the 1980s13. The immunodominant antigen in meningococcal OMVVs is PorA14, an abundant outer-membrane porin with eight surface-exposed loops15. Loops one and four are termed variable region 1 and 2 (VR1 and VR2), respectively, as they generate immune responses and are subject to antigenic variation16. The VR2 loop dominates PorA-specific immunity elicited by OMVVs, which offer limited or no cross-protection against strains expressing PorA with a different VR218. To broaden coverage, OMVVs containing multiple PorAs have been developed21 and selected for the prevalence of PorA sequences in circulating strains22. However, OMVVs present complex manufacturing and regulatory issues23. The recombinant subunit vaccines Bexsero and Trumenba contain an important meningococcal antigen, factor H binding protein (fHbp), which is a lipoprotein composed of two β-barrels that tightly bind domains 6 and 7 of human complement factor H (CFH)27. fHbp is antigenically variable; databases of genome sequences contain more than 900 different fHbp peptides28, which fall into three variant groups or two subfamilies: V1 (subfamily B), V2 and V3 (both subfamily A)30. In general, immunisation with a particular fHbp induces cross-protection against strains that express fHbp belonging to the same, but not a different, variant group, although there can be cross-protection between fHbp variant groups 2 and 3 (subfamily A)32. Bexsero contains a single fHbp peptide (V1.1), with two other recombinant antigens as well as an OMV33, while Trumenba is composed solely of two fHbp peptides (V1.55 and V3.45)34. Antigens in Bexsero and Trumenba have exact sequence matches to 36 and 4.8% of serogroup B N. meningitidis disease isolates currently circulating in the UK, respectively28, leading to concerns about their ability to provide broad coverage against an antigenically diverse pathogen.Two main approaches have been used to develop vaccines against serogroup B s (ChAs) against serogroup B N. meningitidis. ChAs exploit fHbp as a molecular scaffold to present the surface-exposed PorA VR2 loop, which is achieved by inserting the VR2 loop into a β-turn region in fHbp. ChAs retain epitopes from both fHbp and PorA, and can elicit functional immune responses against both antigens. We demonstrate that integration of a VR2 loop does not alter the overall architecture of fHbp and that the VR2 loop folds into a conformation recognised by a bactericidal mAb. We provide proof-in-principle that ChAs can be used to display selected epitopes from integral membrane proteins, such as PorA.Here we use structure-based design to generate chimeric antigen34. fHbp is a lipoprotein that can be expressed as a soluble protein in Escherichia coli following removal of the N-terminal lipobox motif32. As an isolated extracellular loop, the PorA VR2 is likely soluble when expressed separately from the hydrophobic β-barrel of PorA. We exploited soluble fHbp peptide V1.1 as a molecular scaffold to display the PorA VR2 loop, P1.16 (VR2 P1.16). VR2 P1.16 (YYTKDTNNNLTLVP) was inserted into six different β-turn regions in fHbp. At each VR2 P1.16 insertion site, a single amino acid was deleted from the fHbp scaffold  of any β-barrel was 60.5 °C, which is considerably higher than the N-terminal Tm of V3.45 (41 °C), one of the fHbp peptides in Trumenba®  and PorA (H44/76∆fHbp), as well as a H44/76∆fHbp∆porA control. Western blot analyses of lysates from these strains demonstrate that all ChAs elicited antibodies that recognise both fHbp and PorA  as the adjuvant Fig. . Post imorA Fig. , with thHbp Fig. .Fig. 2DeN. meningitidis. We detected antibody binding to fHbp and PorA by flow cytometry, using N. meningitidis strains H44/76∆porA and H44/76∆fHbp, respectively. Results were compared with findings obtained with control sera from mice immunised with phosphate-buffered saline (PBS) and adjuvant alone. Antisera raised against each ChA detected fHbp on the bacterial surface . Significant binding (p ≤ 0.05 by two-way ANOVA) is observed with fHbpV1.1:PorA267/P1.16/MPLA antisera to the H44/76∆fHbp∆porA negative control strain, which is due to non-specific binding  were observed for antisera raised against fHbpV1.1:PorA151/P1.16, fHbpV1.1:PorA117/P1.16, fHbpV1.1:PorA183/P1.16 and fHbpV1.1:PorA309/P1.16 when MPLA, rather than alum, was used as the adjuvant.The serum bactericidal assay (SBA) assesses the ability of antibodies to initiate complement-mediated lysis of 128 Fig. . SignifiN. meningitidis H44/76∆porA; all sets of ChA/adjuvant antisera generated protective SBA titres ≥ 256 , or fHbpV1.1:PorA309/P1.16 when MPLA was the adjuvant . However, significant IgG1 binding to PorA on the surface of H44/76ΔfHbp was observed only with antisera raised against fHbpV1.1:PorA294/P1.16, fHbpV1.1:PorA117/P1.16, fHbpV1.1:PorA183/P1.16 or fHbpV1.1:PorA309/P1.16 with alum  must recruit the C1q subunit of C1dis Fig. . When cores Fig. ; insteadV1.4:PorA151/P1.16, 3.7 Å for fHbpV1.1:PorA294/P1.16 and 2.6 Å for fHbpV1.4:PorA309/P1.16. All ChA scaffolds align with good agreement (route-mean-square deviations (RMSDs) range between 0.447 and 0.614) to fHbp V1.1 , P1.14 (15.2%), P1.9 (12.8%), P1.16 (11.1%) and P1.15  from the integral membrane protein PorA and inserted it into the immunogenic scaffold of the lipoprotein fHbp. We show that multivalent ChAs generate immune responses against two key surface antigens that can elicit protective immunity, providing proof-in principle that immunogenic epitopes from integral membrane proteins can be introduced into a soluble molecular scaffold to create functional antigens, an approach that could be applied to other antigens.During infection pathogens present our immune system with an assortment of surface-exposed, lipid anchored, and integral membrane proteins, all of which can be used as components in subunit vaccines. While lipoproteins are easily engineered for recombinant expression , integral membrane proteins present several challenges for vaccine development. Recombinant forms of integral membrane proteins are often poorly expressed and their native conformations may be compromised during purification, potentially reducing their ability to elicit immune responses against conformational epitopes, such as those found in surface loops35. We found PorA VR2 could induce antibody responses when displayed away from its native environment in the outer membrane. While all ChAs induced antibodies that detected PorA on the surface of N. meningitidis, not all induced bactericidal PorA antibodies. We observed a mixed bactericidal PorA response, which depended on the PorA VR2 peptide, the position of PorA VR2 in the fHbp scaffold and the adjuvant used for immunisation, indicating that several empirically determined parameters are crucial for obtaining bactericidal responses.Combining two antigens within a single recombinant ChA could diminish the immunogenicity of one or both antigens. fHbp is highly immunogenic in all ChAs, inducing bactericidal antibody responses in excess of those correlated with protection47. Structural observations using β-turn peptide mimics demonstrated that cyclised peptides, which mimic their native antigens, are able to induce functional immune responses48. In ChAs, the VR2 N- and C-termini are effectively locked into a 'biological' β-turn peptide mimic by neighbouring fHbp β-strands, as confirmed by our atomic structures. In previous work, cyclic peptides were coupled to carrier proteins and used with adjuvants not licensed for human use46. In the resulting SBAs, titres were only observed with antisera from some mice45, similar to our findings with ChAs and licensed adjuvants.Previous studies found linear VR2 P1.16 peptides failed to elicit antibodies that recognised native PorA, while cyclic VR2 P1.16 peptides, with identical residues fixed in a β-turn, elicited PorA-specific bactericidal antibodies49, while MPLA typically enhances Th1 responses, inducing IgG2a, IgG2b, and IgG350. Murine IgG2a has a greater ability than murine IgG1 to activate the classical pathway51. Consistent with this, ChAs administered with MPLA elicited significantly higher fHbp-specific IgG2a responses and thus higher α-fHbp SBA titres than ChAs administered with alum. Of note, antisera raised against fHbpV1.1:PorA309/P1.16/MPLA had PorA-dependent SBA titre and contained significant levels of α-PorA IgG2a and IgM.Alum allows extended antigen presentation and stimulates T-helper (Th)-2 responses, predominantly producing IgG1 and IgE41 to generate ChAs composed of the most prevalent fHbp and PorA antigens. This maximises vaccine coverage as ChA composition mirrors the prevalent fHbp and PorA antigens circulating within a given geographical area. For example, a vaccine composed of the three most common fHbp peptides from each variant group  with a single PorA VR2 insertion  would give exact sequence coverage against 57% of serogroup B strains circulating in the UK in 2016; this compares favourably with currently licensed meningococcal serogroup B vaccines Bexsero (36%) and Trumenba® (4.8%)41.fHbp and PorA are both antigenically variable. To improve vaccine coverage, we used the comprehensive epidemiology data available for UK meningococcal isolates55, or β-hairpin peptide mimetics57 to develop novel conformationally restricted antigens.In summary, using structure-based design, we generated ChAs that retain epitopes of fHbp and PorA and generate immune responses against both antigens, demonstrating that a soluble antigen can be exploited as a scaffold to display epitopes from an integral membrane protein. Our work provides proof-in-principle for bacterial vaccine design employing structure-led protein engineering previously used in viral proteins to graft functional motifs onto unrelated protein scaffoldsN. meningitidis was grown in the presence of 5% CO2 at 37 °C on Brain Heart Infusion  plates with 5% (v/v) horse serum (Oxoid) at 37 °C. E. coli was grown on Luria-Bertani (LB) agar plates or LB liquid at 37 °C supplemented with 100 μg μl−1 carbenicillin.The bacterial strains used in this study are shown in Supplementary Table fhbp by overlap PCR using the primers in Supplementary Table fHbp by overlap PCR using the primers in Supplementary Table NdeI and XhoI (NEB) and then ligated into pET21b (Novagen); constructs were confirmed by sequencing. Protein expression was performed in E. coli strain B834. Expression cultures were incubated at 37 °C, upon reaching an OD600 of ~0.8, protein expression was induced with 1 mM IPTG. Cultures were harvested after overnight expression at 37 °C. Bacteria were resuspended in Buffer A  and purified by Nickel affinity chromatography  at room temperature. Columns were washed with 25 column volumes (CV) of Buffer A, then 20 CV 80:20 Buffer A:Buffer B ; elution was performed with 10 CV 40:60 Buffer A:Buffer B. Proteins were dialysed overnight at 4 °C into 50 mM Na-Acetate pH 5.5 buffer and further purification was achieved by ion exchange chromatography  at room temperature with a 0–1 M NaCl gradient in 50 mM Na-Acetate pH 5.5 buffer, followed by gel filtration using a HiLoad 16/600 Superdex 75 pg  column equilibrated with PBS (Oxoid).N-terminally truncated fHbp V1.1 was amplified from MC58 genomic DNA using primers fHbp F1 and fHbp R4  from the resulting strains and both mutations were backcrossed into the WT H44/76 background using genomic DNA59.Deletion of N. meningitidis cell lysate60 were probed with one of the following monoclonal antibodies or polyclonal sera: PorA P1.4 mAb , PorA P1.9 mAb , PorA P1.14 mAb , PorA P1.15 mAb , PorA P1.16 mAb , pAb to fHbp V1.160 (diluted 1 in 1000) or polyclonal sera from mice immunised with a ChA (diluted 1 in 500). Following incubation with anti-mouse HRP-conjugated secondary antibodies , membranes were visualised via ECL  on a LAS-4000 (FujiFilm).Western blots of purified proteins (0.5 μg) or 10 μl of 2O by incubating for 5 min in a sonicating water bath. Ten micrograms of MPLA were mixed at room temperature with 20 μg ChA or PBS, 10 mM Histidine-HCl pH 6.5 and 155 mM NaCl. Groups of eight female CD1 mice  were immunised by the intraperitioneal route on days 0, 21 and 35. Four mice were kept in each cage, which were individually vented. Sera was obtained on day 49 following cardiac puncture under terminal anaesthesia. All procedures were conducted in accordance with UK Home Office guidelines. Ethical approval was obtained by the Central Committee on Animal Care and Ethical Review at the University of Oxford. All sera were stored at −80 °C until required; once defrosted, sera were stored at 4 °C.Immunisations were performed with each ChA and PBS controls, using alum or MPLA as the adjuvant. Alum immunisations were prepared by incubating 20 μg ChA or PBS, 2% Alhydrogel (Invivogen), 10 mM Histidine-HCl pH 6.5 and 155 mM NaCl overnight at 4 °C on an end-over-end rocker. For MPLA immunisations, lyophilised MPLA (Invivogen) was resuspended in sterile H60, with the following modifications. N. meningitidis was suspended in Dulbecco’s PBS with cations (Gibco) supplemented with 0.1% glucose (DPBS-G) to a final concentration of 1.25 × 104 CFU ml−1. Baby rabbit complement  was diluted with DPBS-G to a final dilution of 1/10. Serum, pooled or from individual mice, was heat inactivated for 1 h at 56 °C and added to the wells in a serial two-fold dilution, starting with a dilution of 1/5 or higher. Control wells contained no serum or no complement. Following static incubation for 1 h at 37 °C in the presence of 5% CO2, 10 µl from each well was plated onto BHI plates in triplicate and colonies from surviving bacteria counted. Bactericidal activity is expressed as the dilution of serum required to kill≥50% of bacteria in assays containing both complement and serum in comparison with control assays containing serum or complement alone. SBAs using pooled sera were repeated three times, and assays using sera from individual mice were repeated twice. SBA titres were input into GraphPad Prism and statistical analyses comparing titres obtained from alum immunisations with titres obtained from MPLA immunisations were performed using two-way ANOVA  and Dunnett’s method of multiple comparisons.SBAs were performed as previously described6/7 (1–16 nM) were injected over the flow channels at 40 µl min−1). Dissociation was allowed for 300 s. BIAevaluation software was used to calculate the Kd.SPR was performed using a Biacore 3000 . Recombinant ChAs were dissolved in 50 mM sodium acetate pH 4.5 and immobilised on a CM5 sensor chip . Increasing concentrations of CFH−1 and screened for crystal formation via the sitting drop method at a 0.4:0.6 ratio of protein to mother liquor. Crystals of chimeras fHbpV1.4:PorA151/P1.16, fHbpV1.1:PorA294/P1.16, fHbpV1.1:PorA309/P1.16 and fHbpV1.4:PorA309/P1.16 grew under the respective conditions: 2.0 M ammonium sulphate, 0.15 M sodium citrate pH 5.5; 20% (w/v) PEG4000, 0.3 M ammonium sulphate; 0.01 M zinc chloride, 0.1 M sodium acetate, 20% (w/v) PEG6000; and 0.2 M potassium formate, 20% (w/v) PEG3350 respectively. Crystals were transferred to a cryoprotectant solution comprised of mother liquor and 30% ethylene glycol. Data were collected at Diamond light source and integrated/scaled using the Xia2 programme. Molecular replacement was carried out using Phaser from the CCP4 package61, with the fHbp V1.1 structure used as a search model (PDB 2W80). Refmac562, Coot63 and Phenix67 were utilised for rebuilding and refinement, with structural validation performed in Molprobity68. Refinement statistics for each structure are detailed in Supplementary Table ChAs in PBS were concentrated to 7 mg mlTm) is recorded for the fHbp N-terminal and C-terminal β-barrels (Table Purified ChAs (20 μM)  in PBS were subjected to a 20 to 120 °C temperature gradient on a Malvern VP Capillary DSC. Melting temperature  were fixed in 3% paraformaldehyde (PFA) for 1 h. PFA was removed by washing cells three times with PBS. To measure binding of serum antibodies to fHbp and PorA, 1 × 108 cells were incubated for 1 h at 4 °C, shaking at 1250 rpm, with sera diluted 1/50 in PBS. Cells were washed three times with PBS, then incubated for 30 min at 4 °C with shaking at 1250 rpm, with a secondary antibody . Final dilutions of secondary antibodies were as follows: 5 μg ml−1 for Alexa-488 conjugated anti-mouse IgAGM, and 2.5 μg ml−1 for each of Alexa-488 conjugated anti-mouse IgM, IgG3 and IgG2b, and Alexa-647 conjugated anti-mouse IgG2a and IgG1. Cells were washed three times with PBS and then resuspended in PBS for analysis on a FACSCalibur (BD Biosciences). Data were imported into the FlowJoTM data analysis package and transformed to the Logicle scale. A uniform gate was applied to all data sets, based on bacteria visualised by forward and side-scatter, and the geometric mean of fluorescence calculated for FL1H  and FL4H . Each flow experiment was repeated three times, and the FL1H and FL4H geometric means were input into Graphpad Prism. Two-way ANOVA  was used to compare the Alexa-488 or Alexa-647 geometric means with control Alexa-488 or Alexa-647 geometric means. Control results were obtained by incubating PFA fixed cells with sera from mice immunised with PBS and Alum or MPLA adjuvants, and the corresponding Alexa-fluor-labelled secondary Ig. Two-way ANOVA multiple comparisons were performed with geometric means from sets of experiments using the same secondary antibody, N. meningitidis strain and adjuvant; the only variable factor was the ChA used for immunisation.5NQP for fHbpV1.4:PorA151/P1.16; 5NQX for fHbpV1.1:PorA294/P1.16;5NQZ for fHbpV1.1:PorA309/P1.16; and 5NQY for fHbpV1.4:PorA309/P1.16.Structural data that support the findings of this study have been deposited in RCSB Protein Data Bank with the primary accession codes Supplementary InformationPeer Review File


The aim of this study was to investigate the contributions of non-genetic and genetic factors on the variability of stable warfarin doses in Thai patients.VKORC1 − 1639G > A, CYP2C9*3, CYP4F2 rs2108622, and UGT1A1 rs887829 genes were detected from gDNA using TaqMan allelic discrimination assays.A total of 250 Thai patients with stable warfarin doses were enrolled in the study. Demographics and clinical data, e.g., age, body mass index, indications for warfarin and concomitant medications, were documented. Four single nucleotide polymorphisms in the VKORC1 − 1639G > A required significantly lower warfarin stable weekly doses (SWDs) than those with wild-type genotype (p < 0.001). Similarly, the patients with CYP2C9*3 variant allele required significantly lower warfarin SWDs than those with homozygous wild-type (p = 0.006). In contrast, there were no significant differences in the SWDs between the patients who carried variant alleles of CYP4F2 rs2108622 and UGT1A1 rs887829 as compared to wild-type allele carriers. Multivariate analysis, however, showed that CYP4F2 rs2108622 TT genotype accounted for a modest part of warfarin dose variability (1.2%). In contrast, VKORC1 − 1639G > A, CYP2C9*3, CYP4F2 rs2108622 genotypes and non-genetic factors accounted for 51.3% of dose variability.The patients with variant genotypes of VKORC1 − 1639G > A, CYP2C9*3, and CYP4F2 rs2108622 polymorphisms together with age, body mass index, antiplatelet drug use, amiodarone use, and current smoker status explained 51.3% of individual variability in stable warfarin doses. In contrast, the UGT1A1 rs887829 polymorphism did not contribute to dose variability.The online version of this article (doi:10.1007/s00228-017-2265-8) contains supplementary material, which is available to authorized users. The CYP2C9*2 and CYP2C9*3 variants have been shown to have decreased enzymatic activity, which partly accounts for the variance in warfarin dose requirements [CYP2C9*2 or CYP2C9*3 allele required lower warfarin doses than those patients with the CYP2C9*1 allele [VKORC1 gene encodes the VKORC1 enzyme, which is the target enzyme of warfarin action. As reported in several of these ethnic groups, the promoter polymorphism in VKORC1-1639G > A, which is, in turn, associated with reduced VKORC1 messenger ribonucleic acid expression, results in the requirement for a lower warfarin dose [VKORC1-1639G > A or/and VKORC1 1173C > T required significantly lower warfarin doses compared to patients carrying the wild-type allele [Warfarin is a widely used oral anticoagulant for the prevention and treatment of various thromboembolic diseases including atrial fibrillation, heart valve replacement, pulmonary embolism, and deep vein thrombosis . Due to 1 allele –8. The Vrin dose , 9, 10. e allele .CYP4F2 rs2108622 variant allele may have a reduced capacity to metabolize vitamin K and, therefore, require a higher warfarin dose to obtain the same anticoagulant response [CYP4F2 rs2108622 plays a minor role in warfarin dosage requirement in Caucasians and Asians but not African Americans [CYP2C9 and VKORC1, there are a limited number of studies that investigated the effect of CYP4F2 polymorphism on the warfarin dose requirement in Asians. Recently, the polymorphisms of uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1) rs8175347 and rs887829, which are in linked disequilibrium, were significantly associated with a requirement for higher warfarin doses in Korean and Brazilian patients [UGT1A1 rs8175347 and rs887829 variant alleles are classified as poor metabolizers [S-warfarin [S-warfarin. There are few reported data on the association between UGT1A1 polymorphisms and warfarin doses among multi-ethnic patient populations. Therefore, further study is required to elucidate the impact of UGT1A1 polymorphisms on warfarin dose requirements in different ethnic groups.A previous study reported that CYP4F2, a vitamin K oxidase enzyme, was involved in the metabolism of vitamin K to hydroxyvitamin K . The carresponse . The premericans , 16, 17.patients , 19. Thebolizers . Also, 7VKORC1 and CYP2C9, only explain approximately 57–58% of the variability in warfarin dosage. Age, weight, height, smoking and interacting medications accounted for less than 20% of the variance [CYP2C9*3 and VKORC1 on the warfarin dose requirements, only a few studies simultaneously characterized the effects of all drug metabolizing enzymes in the warfarin metabolic pathway and non-genetic factors in the same cohort study. Thus, the objective of this study was carried out to determine whether non-genetic and genetic factors of relevant drug metabolizing enzymes were involved in warfarin metabolism (CYP2C9*3 and UGT1A1 rs887829), site of action (VKORC1-1639G > A), and vitamin K metabolism (CYP4F2 rs2108622).Taken together, non-genetic factors and two main genetic factors, variance . More thA total of 250 patients at The Queen Sirikit Heart Center of Northeast Thailand and Srinagarind Hospital of the Faculty of Medicine, Khon Kaen University who had stable doses of warfarin, were retrospectively recruited from the outpatient clinic during 2015–2016. Indications for warfarin in these patients were atrial fibrillation, heart valve replacement, deep vein thrombosis, pulmonary embolism, or chronic thromboembolic pulmonary hypertension. The inclusion criteria of study subjects included: (1) Thai patients were at least 18 years of age; (2) the international normalized ratio (INR) target range was 2.0–3.0; (3) stable weekly doses (SWDs) of warfarin were defined as the constant dose taken at two consecutive clinical visits for at least 1 month within a target INR range of 2–3; (4) patients who had a measured INR that deviated from the target range by ≤0.2 INR units and that did not result in a change in stable warfarin doses were enrolled in the study. Exclusion criteria included: (1) pregnant patients; (2) patients who had laboratory tests showing abnormal liver function tests ; (3) patients suffering from thyroid disorders. The study was approved by The Khon Kaen University Ethics Committee for Human Research (HE581068). Written informed consent was obtained from all patients.The medical records were reviewed. Demographic variables, i.e., gender, age, height, body weight, and body mass index (BMI) and clinical variables, i.e., indications for warfarin, SWDs of warfarin, INR values, concomitant medications, concomitant diseases, alcohol drinking, and smoking status were recorded. Current age was age at the last monitoring point while other covariates such as height, body weight, BMI, comorbidity, and concurrent medications were noted from the stable warfarin dose period.VKORC1 , CYP2C9*3 , CYP4F2 , and UGT1A1  was performed by the Taqman allelic discrimination assay using a 7500HT Applied Biosystems thermal cycler .Genomic DNAs of study patients were extracted from peripheral blood using QIAamp DNA Blood Mini Kit . Genotyping of p value of less than 0.01 was assumed to indicate a deviation from HWE. Univariate and multivariate analyses were performed to compare the association of non-genetic factors, e.g., demographic and clinical variables and genetic single nucleotide polymorphisms (SNPs) of interest with stable warfarin doses. Associations between stable warfarin doses and age, weight, height, BMI, and mean INR were evaluated using the Spearman’s correlation. The Mann-Whitney U test was used to compare the mean values of warfarin SWDs between two groups. The Kruskal-Wallis H test was used to determine associations between the mean values of warfarin SWDs of the three different genotype groups.Deviations from the Hardy–Weinberg equilibrium (HWE) were tested using a chi-square goodness-of-fit analysis. A p < 0.05 were entered into the multiple linear regression model. In addition, the variables from univariate analyses with p > 0.05  were entered into the multiple linear regression model. Stepwise selections of variables were entered into the regression method including non-genetic and genetic variables that met the default criteria of p < 0.05 for entry and p > 0.1 for removal. A 5% two-tailed significance level was used in all tests, and the p value of <0.05 was considered significantly different. The relative contributions of non-genetic and genetic factors to the total variability of warfarin dose demands were estimated according to the cumulative square of the correlation coefficient (R2). The final R2 of the model indicates the joint contribution of all variables to the variability in therapeutic warfarin dose requirements. The contribution of each factor was calculated as the difference of consecutive cumulative R2 of each variable. All analyses were performed using SPSS 17.0 software .Multiple linear regression analysis was used to investigate the factors that independently affected the inter-individual variability of warfarin dose requirements. The variables from univariate analysis with 2; the average BMI was 24.2 kg/m2. The average of warfarin SWDs was 22.3 ± 9.2 mg. The average INR of all patients was 2.42 ± 0.28. The most common indication for warfarin therapy was atrial fibrillation (67.2%). Over 45% of the study subjects had co-morbid chronic diseases including hypertension, diabetes mellitus, dyslipidemia, and congestive heart failure. From univariate analysis, age, BMI, amiodarone use, antiplatelet drug use, e.g., clopidogrel, prasugrel, and ticagrelor, atrial fibrillation, and deep vein thrombosis/pulmonary embolism/chronic thromboembolic pulmonary hypertension were found to be significantly associated with SWDs of warfarin (p < 0.05) (Supplementary Table p = 0.998)  are summarized in Supplementary Table 2) required a significantly 24.9% higher SWDs of warfarin as compared with normal weight patients (Table 2), overweight (25–29.9 kg/m2), and class III obesity (morbidly obese) (≥ 40 kg/m2) were not significantly different from normal weight patients  consistent with the results of the above-mentioned analyses. In contrast, the stable weekly warfarin doses were significantly positively correlated with body weight , BMI . Height and mean INR had no significant correlations with the stable weekly warfarin doses  (data not shown).The non-parametric Spearman-rank correlation analysis of the data revealed that the stable weekly warfarin doses were significantly negatively correlated with age . The comparisons of warfarin SWDs among those patients with different genotypes are shown in Table VKORC1 − 1639G > A required 49.7% and 27.7% significantly lower averages of SWDS of warfarin as compared with those with wild-type GG genotype (p < 0.001). The averages of warfarin SWDs were 64.6% and 34.1% lower in CYP2C9 *3/*3 and *1/*3 patients than in *1/*1 patients (p = 0.006). The dosage requirement differences, however, were not observed in patients with variant genotypes of CYP4F2 rs2108622 and UGT1A1 rs887829 (p = 0.172 and p = 0.921)  (Table CYP2C9 *1/*3 and CYP2C9 *3/*3 genotypes as compared with those with wild-type *1/*1 genotype (p < 0.001) (Table p < 0.001) (Table p < 0.001 and p = 0.021) (Table CYP4F2 rs2108622 as compared with those with the wild-type CC genotype (p = 0.016) , site of action (VKORC1), and vitamin K metabolism (CYP4F2) on warfarin dose requirements in Thai patients. Apart from VKORC1 and CYP2C9 genes, these results showed that the CYP4F2 gene also contributed to warfarin dose requirements in Thais at a modest level, whereas UGT1A1 did not contribute to warfarin dose variability.Results from the present study suggest that genetic factors, including VKORC1 − 1639G > A and CYP2C9*3 were the major genetic determinants of warfarin dose requirements, with CYP4F2 having a smaller effect [VKORC1 (−1639G > A) AA genotype and 3.7% from VKORC1 (−1639G > A) GA genotype)   [CYP2C9*2 allelic variant and lower frequencies of the CYP2C9*3 were found in Asians as compared with Caucasians [CYP2C9*3 allele in this study. For the CYP4F2 rs2108622 polymorphism, the results revealed that the variant TT genotype was identified as a significant contributor and accounted for a modest part of warfarin dose variability (1.2%)  [UGT1A1 rs887829 SNP was, however, higher in Caucasians (31%) and African-Americans (43%) [UGT1A1 rs887829 in this study population is similar to Koreans, the effects of UGT1A1 rs887829 on warfarin dose requirements between Thais and Koreans were different. The reason for these different results is still not well understood.The contribution of  reports . The pren Asians , which wucasians  which co1.4–4.0% , 24. Acc. 13.2%) . MAF of ns (43%) . AlthougIn terms of non-genetic factors, these results were similar to those previously reported in the literature , 7, 29, VKORC1-1639G > A and CYP2C9*3 were the strongest genetic predictors of warfarin doses in Thai patients, with CYP4F2 rs2108622 having a minor effect; with age, amiodarone use, and BMI being important clinical predictors. Therefore, a more accurate prediction of warfarin dose requirements needs to take into account both genetic and non-genetic factors. There were inherent limitations to the retrospective study design. Confounding factors could not be controlled, e.g., diet containing vitamin K. Prospective studies are needed to determine whether prediction of optimal warfarin doses are enhanced, and adverse effects are avoided by using genetic  and non-genetic factors, e.g., age, amiodarone use, BMI, antiplatelet use, and current smoking status to help guide the selection of a patient’s initial warfarin and maintenance doses.In summary, results in this present study show that genetic and non-genetic factors are important in determining the variability in warfarin response. Supplementary Table 1(DOCX 16 kb)


Csnk1d null embryos, and multiple centrosomes in mouse embryo fibroblasts (MEFs) deficient in CK1δ (MEFCsnk1d null). Results from γ-H2AX staining and the comet assay demonstrated significant DNA damage in MEFCsnk1d null cells. These cells often contain micronuclei, an indicator of genomic instability. Similarly, abrogation of CK1δ expression in control MEFs stimulated micronuclei formation after doxorubicin treatment, suggesting that CK1δ loss increases vulnerability to genotoxic stress. Cellular levels of total and activated checkpoint kinase 1 (Chk1), which functions in the DNA damage response and mitotic checkpoints, and its downstream effector, Cdc2/CDK1 kinase, were often decreased in MEFCsnk1d null cells as well as in control MEFs transfected with CK1δ siRNA. Hydroxyurea-induced Chk1 activation, as measured by Ser345 phosphorylation, and nuclear localization also were impaired in MEF cells following siRNA knockdown of CK1δ. Similar results were observed in the MCF7 human breast cancer cell line. The decreases in phosphorylated Chk1 were rescued by concomitant expression of siRNA-resistant CK1δ. Experiments with cycloheximide demonstrated that the stability of Chk1 protein was diminished in cells subjected to CK1δ knockdown. Together, these findings suggest that CK1δ contributes to the efficient repair of DNA damage and the proper functioning of mitotic checkpoints by maintaining appropriate levels of Chk1.Casein kinase 1 delta (CK1δ) is a conserved serine/threonine protein kinase that regulates diverse cellular processes. Mice lacking CK1δ have a perinatal lethal phenotype and typically weigh 30% less than their wild type littermates. However, the causes of death and small size are unknown. We observed cells with abnormally large nuclei in tissue from  S. pombe [Casein kinase 1 delta (CK1δ) is an evolutionarily conserved serine/threonine kinase that participates in diverse cellular processes, including vesicle trafficking, chromosome segregation, circadian rhythm, Wnt signaling, neurite outgrowth and ciliogenesis . SeveralS. pombe .beta-TRCP ubiquitin-ligase, consequently promoting the stability and function of p53[In mammalian cells, CK1δ localizes to microtubules, the Golgi apparatus, cytoplasmic vesicles and the centrosome as well as the mitotic spindle apparatus , 12, 13,on of p53, 18. ThuCheckpoint kinase 1 (Chk1), a serine/threonine-specific protein kinase, is another primary mediator of the DNA damage response (DDR) and mitotic checkpoints –21. Initin vitro [A recent study showed that Chk1 phosphorylates rat and human CK1δ at the C-terminus, and that site-specific phosphorylation of CK1δ by Chk1 modulates the activity of CK1δ in vitro , indicatDoxorubicin (cat#D1515), hydroxyurea (cat#H8627), DAPI , cycloheximide (cat#C-7698) were purchased from Sigma-Aldrich . PF670462 (cat#3316) and LH846 (cat#4896) were purchased from TOCRIS . Propidium iodide was from Life Technologies (cat#P3566).Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Our protocol was approved by the NCI-Bethesda Animal Care and Use Committee (Protocol Number: LCMB-029). Mice were obtained from The Jackson Laboratory . Pregnant female mice were euthanized via inhalation of carbon dioxide. Isolation of retinal tissue from mouse embryos for immunostaining was performed as previously reported [Csnk1d floxed allele  [Csnk1d null (MEFCsnk1d null) or control MEF (MEFCtl.) cells were also performed as previously described [g for 5 min at room temperature (RT). Upper layer was transferred to new tube, then mixed with 1 ml of 100% ethanol, followed by centrifugation at 20,817 × g for 5 min at RT. Supernatant was discarded and genomic DNA pellet was washed with 70% ethanol by 1 min centrifugation. Genomic DNA was dissolved with 100–200 μl ultrapure water and used for polymerase chain reaction (PCR). PCR protocol indicated at The Jackson Laboratory website was used for genotyping. Briefly, PCR protocol was 95°C for 5 min, 95°C/60°C /72°C (45 sec each) for 35 cycles, followed by 72°C for 10 min and 15°C for infinite time. PCR primer sequences were 9903: GCT GAG GCC AAT AAA GAG TCT GTC ACA TG (common), 9905: GTA GTT TGC CTG ATG AAA CAG GAG C (WT forward), 9906: CAG CCC TAG TTA TCG TAA CAT CG (mutant forward). PCR product size of mutant was 612 bp, heterozygous was 612 bp and 235 bp, wild type was 235 bp.This study was carried out in accordance with the recommendations in the reported . Isolati 010487)  and gene2 humidified 37°C cell culture incubator. MCF7 cells were maintained in RPMI 1640 supplemented with 10% FBS and penicillin/streptomycin.MEF cells and HEK293 cells were maintained in Dulbecco’s modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin (pen/strep) in a 5% CO7 cells/ml. One ml of cell suspension was pipetted into a Falcon 12x75 mm polystyrene tube with snap cap and placed in an ice bath for 15 min. While vortexing the tube on medium speed, 1 ml ice-cold 70% ethanol was added gradually, and samples were stored at 4°C overnight for fixation. Cells fixed in ethanol were spun down by centrifugation , and the ethanol was removed. RNAse  was added to each tube and incubated at 37°C for 20 min, followed by the addition of 500 μl propidium iodide (50 μg/ml in PBS). Cells were incubated at 4°C in the dark for at least 30 min before flow cytometry. Cell cycle analysis was performed with BD Calibur at NCI FACS core facility (https://ostr.cancer.gov/resources/ncr/Flow%20Cytometry) using BD CellQuest software (BD Biosciences) and ModFit software .Cells were grown in culture dishes, collected and resuspended in phosphate-buffered saline (PBS) at a concentration of 1–2 x 10® Kit  according to the manufacturer’s protocol. Briefly, Comet LM Agarose  and cells  were combined, and 50 μl of the mixture was immediately placed onto a comet slide. After 30 min incubation at 4°C in the dark, slides were immersed in 4°C lysis solution for 2 h at RT. Subsequently, slides were immersed in freshly prepared alkaline unwinding solution (pH > 13) for 1 h at RT in the dark. Using a CometAssay™ ES unit, electrophoresis was performed with Alkaline Electrophoresis Solution at 21 volts for 1 h. After draining the excess electrophoresis solution, slides were immersed twice in dH2O, once in 70% ethanol for 3 min, and then dried at 37°C for 15 min. SYBR Green solution  was added to the dried agarose samples, which were stained for 30 min at RT in the dark. The comet image was captured with epifluorescent microscopy and tail moment was analyzed with OpenComet software (http://www.cometbio.org).The comet assay was performed with CometAssayCtl. cells were seeded at 5,000 cells/well in 96-well plate on the day before treatment. PF670462-mediated cytotoxcity was assessed by the MTS assay using Cell Titer 96® AQueous One Solution Cell Proliferation Assay  according to the manufacturer’s protocol  according to the manufacturer's protocol. For CK1δ knockdown in MEF cells, Mm_Csnk1d_1 FlexiTube siRNA  and Mm_Csnk1d_2 FlexiTube siRNA  were used. For CK1δ knockdown in human cells, Hs_CSNK1D_6 FlexiTube siRNA  and CSNK1D siRNA from Ambion (siCK1δ_Am) were used  and a kinase-dead (K38A) derivative were previously described , 6. For See g for 15 min at 4°C. Supernatant and pellets were separated, and supernatant was used for detection of other proteins , while pellets were resuspended with 15 μl of 0.2N HCl and incubated on ice for 15 min. Pellet samples were neutralized with 2 μl of 2M NaOH, then resuspended with lysis buffer and protein concentration was determined  prior to SDS-PAGE and immunoblotting for γ-H2AX.Eighty to 90% confluent monolayers that had been seeded in 6- or 12-well cell culture plates were rinsed twice with PBS, lysed and processed for SDS-PAGE and western blot analysis as previously described . For immHEK293 cells were harvested with IP buffer , supplemented with protease inhibitors . Cell lysates were homogenized by passing through 23-gauge syringe needles, and centrifuged at 20,817 × g for 15 min at 4°C. Supernatant was transferred to another tube, and protein concentration was determined by Bio-Rad protein assay dye reagent. Cell lysates (1 mg) was incubated with 1 μg of mouse IgG and 20 μl of protein A/G Plus-agarose for 2 h at 4°C, followed by centrifugation at 20,817 × g for 10 min. In parallel, 1 μg of FLAG antibody and 20 μl of protein A/G-agarose were combined in 1 ml IP buffer and incubated for 2 h at 4°C with rotation. After 2 h, centrifugation at 956 × g for 5 min. Pre-cleared lysate and target antibody/protein A/G-agarose mixture were combined and incubated overnight at 4°C with rotation. After 18 h, protein A/G beads were washed with IP buffer three times, and 30 μl of 2x Laemmli buffer (supplemented with 5% beta-mercaptoethanol) was added. The samples were heated at 95°C for 10 min, and subjected to SDS-PAGE followed by western blotting.2, pH 6.9). Cells were fixed with freshly prepared 3.7% formaldehyde and 1% Triton X-100 in PHEM supplemented with Taxol (10 mM) for 10 min at RT. Cells were then washed with PBS, and blocked with 6% BSA in PBS for 30 min at RT. Then primary antibody/antibodies was/were added with blocking solution, and samples were incubated for 1 h at 37°C or overnight at 4°C. After washing three times with PBS, samples were incubated with secondary antibody reagent(s) and DAPI in blocking solution for 45 min at RT. After washing three times with PBS, coverslips were mounted on glass slides (VWR Scientific) using ProLong Gold Antifade reagent . To visualize gamma-tubulin, cells were first washed once with PBS, then with PHEM, followed by treatment with PHEM containing 0.19 M NaCl, 1% Saponin, 10 μM Taxol, and 0.1% DMSO for 5 min at RT to permeabilize cells and stabilize tubulin. Cells were then immersed in cold 100% MeOH at −30°C for 10 min, rehydrated by rinsing three times with PBS, and treated with blocking solution for 30 min at 37°C. Primary and secondary antibodies were added as described above.MEF cells cultured on 12 mm diameter glass coverslips  were first washed once with PBS, then once with PHEM .Fluorescent images were collected with a laser-scanning confocal microscope (510 LSCM), and a 63x objective (Carl Zeiss Inc.). Zeiss LSM image browser Version 4.0.0.157 was used for image processing, and composite figures were prepared with Adobe PhotoShop CS3 Ver 10.0.1 (Adobe Systems Inc.). Fluorescence intensity was measured by ImageJ software  was added at a final concentration of 10 μg/ml to the cells for 8, 6, 2 and 1 h treatment, respectively. Forty-eight h after siRNA transfection, cells (including ones that did not receive CHX) were harvested and western blot analysis was performed.MEFt-test unless otherwise noted. The differences were considered to be significant when P value was less than 0.05. For B in The significance of differences in data was determined with Student’s Csnk1d allele previously shown to disrupt expression of CK1δ protein have a perinatal lethal phenotype, and weigh 30–50% less than heterozygous littermates [Csnk1d null mice [Csnk1d null embryos often had grossly enlarged nuclei  of cells with large, DAPI-stained nuclei that contained three or more centrosomes  Csnk1d alull mice , we notid nuclei . Analysitrosomes , featurel. cells . Micronull cells . MicronuCtl. cells in monolayer culture showed signs of a DDR in the absence of exposure to a genotoxic agent. In marked contrast, MEFCsnk1d null cells exhibited massive γ-H2AX staining, indicating that a lack of CK1δ induced a strong DDR  in cells deficient in CK1δ by immunostaining with γ-H2AX antibody . Only a rong DDR . A similrong DDR , a humanl. cells . Substanl. cells . Treatmeiability . This imCsnk1d null cells stained positively for LC3 (microtubule-associated protein 1A/1B-light chain 3), which is recruited to autophagosomal membranes during the process of autophagy  CK1δ expression vector partially restored Chk1 activation in response to HU  normally stimulates replicative stress and activates Chk1, via phosphorylation at Ser345 . The timl. cells . Furtheron by HU . A correon by HU . Importase to HU . Transfese to HU . To furt of Chk1 . This reCtl. cells treated with CK1δ or negative control siRNA. Typically, Chk1 localizes to the cytoplasm and nucleus; retention in the nucleus requires Ser345 phosphorylation [To further evaluate the impact of CK1δ on Chk1 function, we examined the intracellular distribution of Chk1 in MEFrylation . As expeCtl. cells were transfected with either control or CK1δ siRNA, and then treated with cycloheximide (CHX). The Chk1 protein level was intact for at least 6 h after CHX treatment in cells that received the control siRNA. In contrast, the Chk1 protein level decreased significantly in MEFCtl. cells transfected with CK1δ siRNA S7 Fig). The reduced amount of total and phosphorylated Cdc2/CDK1 in cells lacking CK1δ also raises mechanistic as well as functional questions. Cdc2/CDK1 turnover during genotoxic stress has been attributed to polyubiquitination following its phosphorylation by double stranded RNA-activated protein kinase , 50. We Other members of the CK1 family have been implicated in the regulation of Chk1 activation and Cdc2/CDK1 phosphorylation. CK1γ1 phosphorylation of Claspin, a binding partner of Chk1, is critical for Chk1 activation, but CK1γ1 has little or no effect on total Chk1 protein level . CK1α phCsnk1d null embryo showed signs of microcephaly Click here for additional data file.S2 FigCtl. cells and MEFCsnk1d null cells. Red peaks correspond to G0/G1 and aqua peaks correspond to sub G0/G1 region. The latter is comprised of dead cells or cell fragments with markedly reduced amounts of DNA.Cell cycle analysis was performed with PI staining using different passage number of MEF(PDF)Click here for additional data file.S3 Figp<0.05. (C) Cells that had been treated as described in (A) were immunostained with DAPI and γ-H2AX antibody. Bars, 20 μm. (D) Relative fluorescence intensity of γ-H2AX. Six images in each group  were analyzed with Image J software. **p<0.01, ***p<0.001.(A) Cells were transfected with negative control siRNA (siNeg) or two different siRNAs targeting CK1δ (siCK1δ Q6 and siCK1δ Am). 72 h later, cells were collected and lysates were immunoblotted for γ-H2AX. Panel represents one of three independent experiments. (B) Relative band intensity of γ-H2AX normalized to loading control. Data are presented as the mean plus standard deviation of three experiments. *(PDF)Click here for additional data file.S4 FigCtl cells were treated with PF670462 (10 μM) for 5 hours. Scale bar = 10 μm. (B) Micronuclei formation was induced by PF670462 treatment in MEFCtl cells. Incidence of micronuclei was measured in control (DMSO) and PF670462 treated groups; 50 and 54 cells were counted respectively, and statistical analysis was performed with Fisher’s exact test. ***p<0.0001. (C) Cell viability was tested with MTS assay. MEFCtl cells were treated with PF670462 for 3 days. Data is shown as average of 4 independent experiments with mean +/- SD. **p<0.01, ***p<0.001 compared with DMSO control.(A) MEF(PDF)Click here for additional data file.S5 Fig(A) Cells were transfected with negative control or CK1δ siRNA and treated with HU for the indicated times. Cell lysates were immunoblotted as indicated. (B) Bar graph shows the relative intensity of bands in (A) that were normalized to HSC70. Data are from one representative experiment of multiple experiments.(PDF)Click here for additional data file.S6 FigCtl cells were pre-incubated with the indicated concentrations of PF670462 or LH846 for 1 h, subsequently treated with HU for 1.5 h and harvested for western blotting.MEF(PDF)Click here for additional data file.S7 FigHEK293 cells were transfected with FLAG-Chk1 and various Myc-CK1δ derivatives  . 48 h la(PDF)Click here for additional data file.S8 FigCsnk1d null embryo #A: The cranial vault is greatly expanded compared to the WT. The brain appeared compressed both dorsally and ventral. Throughout the midbrain and brainstem and in the cortical plate are foci of hemorrhage and necrosis. The subventricular zone in the forebrain appears thickened and disorganized compared to WT. The 4th ventricle, aqueduct and lateral ventricle are more dilated than in the WT. Csnk1d null embryo #B: Possible mild compression compared to the WT. In the forebrain, possible increased streaming of subventricular cells into the intermediate zone.(PDF)Click here for additional data file.S9 FigArea 1 shows pontomedullary/medullary hindbrain, and Area 2 shows midbrain stained with H&E. Note that at higher magnification, cells were detected in Csnk1δ null embryos with large cell/nuclear size and abnormal cell shape compared with cells in WT tissue.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Compared to +5°C, CO2 decreased 3- and 10-fold at -5 and -20°C, respectively. High 13C recovery in CO2 from the C-1 position indicates dominance of the pentose phosphate pathway at +5°C. In contrast, increased oxidation of the C-4 position at subzero temperatures implies a switch to glycolysis. A threefold higher 13C recovery in microbial biomass at -5 than +5°C points to synthesis of intracellular compounds such as glycerol and ethanol in response to freezing. Less than 0.4% of 13C was recovered in DOC after 1 day, demonstrating complete glucose uptake by microorganisms even at -20°C. Consequently, we attribute the fivefold higher extracellular 13C in soil than in microbial biomass to secreted antifreeze compounds. This suggests that with decreasing temperature, intracellular antifreeze protection is complemented by extracellular mechanisms to avoid cellular damage by crystallizing water. The knowledge of sustained metabolism at subzero temperatures will not only be useful for modeling global C dynamics in ecosystems with periodically or permanently frozen soils, but will also be important in understanding and controlling the adaptive mechanisms of food spoilage organisms.Although biogeochemical models designed to simulate carbon (C) and nitrogen (N) dynamics in high-latitude ecosystems incorporate extracellular parameters, molecular and biochemical adaptations of microorganisms to freezing remain unclear. This knowledge gap hampers estimations of the C balance and ecosystem feedback in high-latitude regions. To analyze microbial metabolism at subzero temperatures, soils were incubated with isotopomers of position-specifically  Microbial processes in high-latitude ecosystems with permafrost or frozen soils during winter periods are important contributors to global carbon (C) and nitrogen (N) cycling. Metabolic activity has been detected in soil at temperatures as low as -39°C  and an aMost of the studies on metabolism at low temperatures are based on isolated pure cultures from permafrost or used permafrost samples with inherently adapted microbes . However13C incorporated from specific glucose positions into soil, microbial biomass, dissolved organic carbon (DOC), and CO2 were conducted to identify microbial metabolic adaptations in frozen soil habitats. Poor water availability and low thermal energy limit microbial activity in frozen conditions. To survive these stresses, microorganisms form biofilms composed of exopolysaccharides (EPS). Sugars units forming the EPS are synthesized via the pentose phosphate pathway. Therefore, we hypothesized that the glucose C-1 position will be preferentially oxidized to meet energy demands. Consequently, high 13C recovery from the glucose C-1 position is expected in CO2. More of the remaining glucose C positions will be used for EPS synthesis than cellular compounds at subzero temperatures. This would result in higher 13C in the extracellular environment than in microbial biomass from the remaining glucose C positions due to the secreted EPS.Analyses of n = 3) used to determine dry weight. Mean annual temperature and precipitation at the site are +7°C and 874 mm, respectively. The soil had a pH (KCl) of 4.88, a pH (H2O) of 6.49, a total organic carbon (TOC) and total nitrogen content of 1.77 and 0.19%, respectively, and a cation exchange capacity of 13 cmolC kg-1.The soils were collected (0–10 cm depth) from agriculturally used loamy Luvisol in northern Bavaria  in August 2014, sieved to 2 mm, and stored for 1 day at +5°C except for samples , uniformly 13C-labeled (U-13C) and unlabeled glucose  were applied to the soils in separate microcosms with four replicates of each. 5 ml of 2.55 mM glucose solutions were applied on top of the soil. This C concentration was less than 5% of microbial biomass C and therefore, does not alter microbial community structure (2. 20% (w/v) of NaCl was dissolved in NaOH traps at -20°C to depress the freezing point. The microcosms were sealed and incubated at the respective temperatures. NaOH in the vials was exchanged after 10 h, 1, 2, 3, 6, and 10 days at +5 and -5°C and in addition after 15, 20, and 30 days at -20°C. Soil samples were collected after 1, 3, and 10 days at +5 and -5°C and also after 30 days at -20°C, to account for the slow processes expected in deeply frozen conditions. Thirty grams of each sample was immediately subjected to chloroform fumigation-extraction, as described below.Screw-cap glass microcosms (10 cm diameter and height of 12 cm) with a base layer of quartz sand were used for incubations. Eighty grams samples of soil were transferred to sample rings and installed on ceramic plates above the quartz sand. The soils were rewetted to field capacity by adding 10 ml of water to the underlying sand at +5°C for 2 days. Thereafter, the microcosms were preconditioned at +5, -5, and -20°C for 24 h. These temperatures were chosen to simulate (1) average annual soil temperature at the sampling site (control), (2) average winter temperature, and (3) lowest temperatures in some winters on this site and common deep freezer storage temperatures, respectively. Four position-specifically tructure . Cups wi2 trap was diluted 1:10 with ultrapure water, and the CO2 content was determined with a non-dispersive infrared (NDIR) gas analyzer . The remainder of the CO2 in the NaOH traps was precipitated with 5 ml of 0.5 M SrCl2 solution. SrCO3 precipitates were separated by centrifuging four times at 2000 × g for 10 min and washing in between with Millipore water until pH 7 was attained. δ13C values of the dried SrCO3 (1–2 g) were measured with a Flash 2000 elemental analyzer coupled by a ConFlo III interface to a Delta V Advantage isotope ratio mass spectrometer . 13C respired from the applied glucose was calculated according to a mixing model Eqs 1 and 2 (BG) in Eq. 1 was determined by Eq. 2.0.4 ml of each CO 1 and 2 , where twhere:2/BG/appGCO C content of the sample/background/applied glucose (mg C g-1 soil)[C]2/BG/appGCO13C atom %-excess of labeled sample/background/applied glucose (at %)r13C isotope measurements were performed with EuroVector elemental analyzers  coupled by a ConFlo III interface to a Delta Plus XP IRMS . C recovery from applied glucose was calculated according to Eqs 1 and 2.Aliquots of soil were freeze dried, ground in a ball mill and 13–15 mg were weighed into tin capsules. 2SO4 was used to extract organic C on an orbital shaker for 1.5 h. Samples were centrifuged for 10 min at 2000 rpm and the supernatant was filtered for determination of C concentration . About 25 mg (fumigated) and 40 mg (unfumigated) freeze-dried extracts were used for δ13C determination via EA-IRMS, performed by the same instrument coupling used for bulk soil δ13C determination. Incorporation of glucose C into fumigated and unfumigated samples was calculated according to Eqs 1 and 2. Microbial biomass C and 13C were calculated by subtracting unfumigated from fumigated values and dividing by an extractability correction factor of 0.45 . If assumptions of normality and homogeneity of variances with groups were not met, outcomes were validated by a non-parametric Kruskal–Wallis ANOVA. Significant differences were determined with Tukey’s honest significance difference (Tukey’s HSD) post hoc test at a confidence level of 95%. Statistical tests were performed with Statistica .A Nalimov outlier test with 95% significance level was performed on CO2 was released down to -20°C, although cumulatively, there were 3- and 10-fold reductions at -5 and -20°C, respectively, compared to +5°C at day 10. Similarly, recovery of glucose-derived 13C in CO2 at -5 and -20°C was respectively 19 and 24% lower than +5°C . At +5°C, 13C recovery in CO2 showed preferential oxidation of the glucose C-1 position. In contrast, the glucose C-4 position was the most respired at subzero temperatures. Irrespective of temperature, two phases of glucose C mineralization were observed: (1) high glucose 13C respiration within the first 3 days at 5°C and 2 days at -5 and -20°C and (2) low respiration of glucose-derived 13C thereafter .CO13C recovery in bulk soil from uniformly labeled glucose was over 24% higher at -5°C than at +5 and -20°C. After the first day, recovery did not differ with temperature . Similarly, position-specific patterns of 13C recovery in bulk soil were comparable between +5 and -20°C but differed at -5°C. At +5 and -20°C, 13C recoveries in bulk soil from C-2, C-4, and C-6 were significantly higher (p < 0.05) than C-1 . In contrast, recovery from C-2 and C-6 was significantly higher (p < 0.05) than from C-1 and C-4 at -5°C .At day 1, 13C recovery from microbial biomass was obtained at -5°C and it was two and three times higher than at +5 and -20°C, respectively . The position-specific patterns of 13C incorporation into microbial biomass were not affected by temperature. The 13C recovery in microbial biomass from C-1 was ≈1.7 times lower than C-2, C-4, and C-6 at each temperature . Moreover, recovery from each position did not differ over time at any incubation temperature.From uniformly labeled glucose, the highest 13C recovery in bulk soil to microbial biomass was highest at -20°C (factor 3), followed by +5°C (factor 1.5) and -5°C (factor 0.6) .The ratio of extracellular Figure 3A). Whereas DOC did not differ over time at -20 and +5°C, it increased by a factor of 1.5 between day 3 and 10 at -5°C, to reach a level similar to +5°C. 13C recovery in DOC at -20°C was also two times higher than both at -5 and +5°C . Less than 0.4% of the applied 13C was recovered in DOC on day 1, irrespective of temperature . The position-specific 13C patterns detected in CO2 were different to DOC at each temperature. Whereas recoveries in DOC at -5°C did not differ between the four glucose positions, the recovery from the glucose C-6 position was twice as high as C-1 at +5 and -20°C .Total extractable C in soil was highest at -20°C, being 1.4- and 2.3-fold higher than +5°C and -5°C, respectively  suggesting that all applied glucose was taken up by microorganisms within the first day. These results agree with previous data showing that substrate uptake of highly available substances, such as glucose, is largely independent of temperature . The results agree with studies showing strong temperature sensitivity of CO2 production in frozen soils , reflects mineralization of glucose-derived metabolites , and revealed that glucose was predominantly catabolized via the pentose phosphate pathway . High 13C recovery from the C-4 position reflects glucose transformation via glycolysis . In a recent study, 13C incorporation into microbial DNA was not detected after 1 month at subzero temperatures  observed at -5°C .eratures . This sueratures . Hence, eratures , microoreratures . Glyceroeratures . The synydration . Glyceroydration . Glyceroeratures . We emph13C incorporation into microbial biomass at -20°C  at -20°C . This high ratio could not be attributed to the non-metabolized glucose that remained in the soil because less than 0.4% of applied 13C was recovered in DOC at day 1 ; moreover, extracellular 13C recovery increased again at later time points. The high 13C recovery in bulk soil compared to microbial biomass at -20°C suggests that microorganisms utilized glucose for extracellular antifreeze compounds. To resist physical, environmental, and biological stress, and to survive in diverse ecological niches, microorganisms form biofilms. These biofilms are mainly composed of EPS secreted by microbes. Recently, culture-based studies revealed EPS secretion at -15°C by bacterial cells . Increasing glucose concentration was found to increase EPS production at low temperatures . This would account for the higher extracellular 13C in soil at -20°C compared to -5 and +5°C . The poor extractability of EPS in water . This is consistent with the results of both Figure 3A), suggesting that underlying adaptation strategies are rather general. The spectroscopic properties of DOC at -13°C did not indicate lysis of microbial biomass induced by freezing . Increased pentose phosphate pathway activity signifies increased NADPH production, which is required as a source of reduction equivalents in protein and lipid synthesis .In addition to EPS, organisms commonly synthesize AFP to overcome freezing effects. These AFP have been identified in fungi, bacteria, plants, insects, and polar fishes . Some baynthesis . MoreoveThe position-specific tracing of metabolic pathways in our study revealed that microorganisms induce a suite of temperature-dependent physiological processes to cope with freezing. This enables both catabolic and anabolic processes. These physiological changes reflect microbial resilience, enabling them to inhabit any ecological niche. Whether these responses are accompanied by a microbial community shift or solely reflect phenotypic plasticity remains to be determined.13C labeling proved to be a valuable tool in understanding the survival strategies of microorganisms at subzero temperatures. Glucose was metabolized by the pentose phosphate pathway at +5°C and this shifted to glycolysis at subzero temperatures. High 13C recovery within microbial biomass at -5°C suggests that microorganisms invest energy and resources in intracellular antifreeze metabolites. In contrast, strategies differ greatly at -20°C, where predominantly extracellular secretion protects the cells.Position-specific 2 emission (High-latitude C and N dynamic models such as Simple Biosphere/Carnegie-Ames-Stanford Approach (SiBCASA) focuses on extracellular parameters and only the thawing of permafrost is assumed to increase COemission . This inThe concept was contributed by YK and MD, experimental design by EB and MD, data acquisition and analysis by CA and SH, interpretation and drafting by EB, and revision including final approval of the version by YK and MD.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.


Pd, which is 6.88 times higher than commercial Pd/C (0.72 A per mgPd), by shortening the distance between Pd and Ni active sites, achieved through shape transformation from Pd/Ni–P heterodimers into Pd–Ni–P nanoparticles and tuning the Ni/Pd atomic ratio to 1:1. Density functional theory calculations reveal that the improved activity and stability stems from the promoted production of free OH radicals (on Ni active sites) which facilitate the oxidative removal of carbonaceous poison and combination with CH3CO radicals on adjacent Pd active sites.Incorporating oxophilic metals into noble metal-based catalysts represents an emerging strategy to improve the catalytic performance of electrocatalysts in fuel cells. However, effects of the distance between the noble metal and oxophilic metal active sites on the catalytic performance have rarely been investigated. Herein, we report on ultrasmall (∼5 nm) Pd–Ni–P ternary nanoparticles for ethanol electrooxidation. The activity is improved up to 4.95 A per mg Incorporating oxophilic metals into noble metal catalysts can improve electrocatalytic performance; however, the influence of the distance between noble metal and oxophilic metal active site is not well understood. Here the authors make Pd–Ni–P nanocatalysts for ethanol oxidation, with improved performance achieved by shortening the Pd–Ni distance. M) such as gold (Au), silver (Ag) and especially nonprecious 3d transition metals111213151617181920212223Direct fuel cells have been recognized as a promising future power source due to advantages, including environmental aspects, facile storage, easy refilling and high power density123456234589M–P ternary phosphide nanoparticles (NPs)262743CO radicals on the adjacent Pd active sites to generate acetate ions, and this combination has been confirmed as the rate-determining step for EOR10115202930Compared with Pt-based catalysts, palladium (Pd) is more efficient for ethanol oxidation reaction (EOR) in alkaline media due to its relatively high catalytic activity, lower cost and better resistance to CO poisoning72, Ni(acac)2 and trioctylphosphine, and then treated at 290 °C for 1 h. The NPs with a Ni/Pd atomic ratio of 1:1 show the best catalytic performance towards EOR. By tuning the phosphorization temperature and time, the NPs are transformed from Pd/Ni–P heterodimers into Pd–Ni–P NPs with closer Pd–Ni distance , respectively.Herein we report a two-step solvothermal strategy for the synthesis of small (∼5 nm) Pd–Ni–P ternary NPs with tunable Ni/Pd atomic ratio and controlled distance between Pd and Ni active sites (termed as Pd–Ni distance). Briefly, the NPs are synthesized via thermolysis  of the mixture containing Pd(acac)distance . Besides38Ni49P13, 5.5±1.0 nm) to 38Ni45P17, 5.3±1.0 nm) and 40Ni43P17, 5.3±0.5 nm). Different from the reported phosphides using NaH2PO2 and NaBH4/N2H4 as precursors26273238Ni45P17 NPs. It is noteworthy that if the NPs were treated with one-step strategy , the particle size would increase and the size distribution would become wider. Therefore, we used the two-step strategy to fabricate the NPs with different Pd–Ni distance . According to the clear lattice fringes shown in the HRTEM image, the Pd species still existed as large domains in the Ni–P matrixes  analysis detailed later. In comparison, the NPs with only 0.4% of Pd doping show very good crystallinity (12P5 (JCPDS: 22-1190). Therefore, the amorphous structure of our NPs can be attributed to the Pd-doping that causes the lattice distortion.As shown in the transmission electron microscopy (TEM) images , all thematrixes . If the ecreased . Correspallinity , which cThe spatial distribution of Pd, Ni and P species in the Pd–Ni–P ternary NPs was further confirmed via high-angle annular dark-field image and elemental mapping . As show38Ni49P13, Pd38Ni45P17, Pd40Ni43P17 and commercial Pd/C catalysts in the deaerated NaOH solution (1.0 M) at a scan rate of 100 mV s−1. The ECSA (m2 per gPd) values of these nanocatalysts are estimated according to the equation ECSA=Q/(0.405 × WPd), where Q and WPd are the coulombic charge by integrating peak area of the reduction of PdO (mC) and Pd loading (mg cm−2) on the electrode, respectively. Meanwhile, 0.405 represents the charge required for the reduction of PdO monolayer (mC per cm2Pd). Herein, the ECSA values of these Pd38Ni49P13, Pd38Ni45P17 and Pd40Ni43P17 NPs are 56.74, 57.36 and 63.22 m2 per gPd, respectively. Interestingly, the Pd40Ni43P17 NPs demonstrate the largest ECSA, which is 1.33 times higher than that of commercial Pd/C (47.50 m2 per gPd). As expected, the ECSA, i.e., the activity significantly increased with the decrement of the distance between Pd and Ni active sites.The catalytic performance of the as-prepared Pd–Ni–P NPs was investigated and the results were compared with those of commercial Pd/C catalysts. As an important parameter for the assessment of active sites of catalysts, the electrochemically active surface area (ECSA) was first evaluated. The ECSA of these NPs can be calculated from the charge required for oxygen desorption, that is, from the area of the reduction peak of PdO in the cyclic voltammograms (CVs) in 1.0 M of NaOH40Ni43P17 NPs initially enhanced with the increase of cycle numbers as previous report of amorphous electrocatalyst2H5OH at 100 mV s−1. 38Ni49P13 (4.12 A per mgPd), Pd38Ni45P17 (4.42 A per mgPd) and Pd40Ni43P17 (4.95 A per mgPd) NPs are almost 5.72, 6.14, and 6.88 times higher than that of commercial Pd/C (0.72 A per mgPd). Clearly, the Pd40Ni43P17 NPs have the highest mass catalytic activity among these three nanocatalysts at a certain potential (0.8 V). Furthermore, the Pd40Ni43P17 NPs show much slower current decay over time than Pd38Ni49P13, Pd38Ni45P17 NPs and commercial Pd/C (40Ni43P17 NPs (215.4 mA per mgPd) still maintain 5.36 times of commercial Pd/C (40.21 mA per mgPd) electrocatalysts. Moreover, further extending to 20 h, still Pd40Ni43P17 NPs displayed better mass activity than that of Pd/C . After further phosphorization , the ECSA values of Pd54Ni30P16, Pd40Ni43P17 and Pd32Ni50P18 NPs are 37.51 m2 per gPd, 63.22 m2 per gPd and 51.76 m2 per gPd, respectively. As shown in 40Ni43P17 NPs demonstrate the largest ECSA, which is 1.33 times higher than that of commercial Pd/C. All the results suggest that the Pd/Ni ratio around 5/5 in the salt precursor is preferable for good electrocatalytic performance.The catalytic performance of the as-prepared Pd–Ni–P NPs was investigated and the results were compared with those of commercial Pd/C catalysts. As shown in 2H5OH at 100 mV s−1. 47Ni36P17 (4.09 A per mgPd), Pd38Ni45P17 (4.42 A per mgPd) and Pd31Ni53P16 (2.74 A per mgPd) NPs are almost 5.68, 6.1 and 3.81 times higher than that of commercial Pd/C (0.72 A per mgPd). Obviously, the Pd38Ni45P17 NPs have the highest mass catalytic activity among the precursor NPs at a certain potential. The potential was held at 0.8 V during the measurements. Furthermore, the Pd38Ni45P17 NPs show much slower current decay over time than Pd47Ni36P17, Pd31Ni53P16 NPs and commercial Pd/C , Pd40Ni43P17 (4.95 A per mgPd) and Pd32Ni50P18 (3.90 A per mgPd) NPs is always much higher than those of the corresponding Pd47Ni36P17 (4.09 A per mgPd), Pd38Ni45P17 (4.42 A per mgPd) and Pd31Ni53P16 (2.74 A per mgPd) NPs, respectively (40Ni43P17 (4.95 A per mgPd) NPs, which is 6.88 times higher than that of commercial Pd/C (0.72 A per mgPd), further demonstrating that the Pd40Ni43P17 NPs own significantly enhanced electrocatalytic activity. Moreover, the Pd40Ni43P17 NPs show a much better durability for ethanol oxidation than all other NPs and commercial Pd/C  and 3d3/2 (340–342 eV) peaks were observed (5/2 peaks of Pd(0) in Pd38Ni45P17 and Pd40Ni43P17 NPs are located at 335.67 and 335.45 eV, respectively, and both of them positively shift as compared with pure Pd (0) (335.2 eV) reported previously5/2 peak of Pd(0) in Pd40Ni43P17 is negatively shifted 0.22 eV as compared with that in Pd38Ni45P17 NPs , NiO (855.8 and 873.4 eV) and Ni (852.5 and 869.6 eV) 2 apart from a few nominally reduced Ni species. Significantly, after further phosphorization, the Ni 2p3/2 (852.5 eV) in Pd40Ni43P17 NPs is hardly observed, which can be attributed to the formation of Ni–P38Ni45P17 NPs could be assigned to the oxidized P2O5 and P(0) species, respectively, as reported in the literature2838Ni45P17 and Pd40Ni43P17 NPs shifts negatively by 0.7 and 0.8 eV, respectively, with respect to that of red phosphorus40Ni43P17 NPs becomes more negative. These negative shifts in binding energy may be explained by assuming that P(0) species accepts partial electrons from surrounding Pd and Ni26To investigate the mechanism of the satisfied electrochemical catalytic performance, the chemical states of Pd, Ni and P elements in both Pdanalysis . High-reanalysis  indicateobserved . These p5P17 NPs . The pos69.6 eV) . Obvious5/2 334–3 eV and 35/2 334–3 eV and 3ads and COads 132 (99.99%) for 30 min. For simplicity, the weak CO anti-poisoning results of dumbbell Pd38Ni49P13 NPs were not shown here. 38Ni45P17 , indicating an increased CO oxidation activity and good CO anti-poisoning ability of these NPs433ads40Ni43P17 NPs is larger than that of Pd38Ni45P17 and commercial Pd/C, which is in agreement with the electrocatalysis results of EOR. All the results further suggest that the Pd40Ni43P17 NPs demonstrate excellent electrocatalytic performance for EOR and superior CO tolerance ability due to the shortened Pd–Ni distance.To further study the mechanism of good EOR performance, CO anti-poisoning experiments were also carried out. Chemisorbed CO intermediate has been identified as a major poison species for EOR on the active sites of catalysts398Ni45P17  and Pd403P17 NPs  recordedM) surfaces in alkaline media proceeds via the reactive-intermediate and/or the poisoning-intermediate (CO) pathway (10M-OHads (*OH) and M-(COCH3)ads (*CH3CO) intermediates are combined to generate acetate anions. This combination between *OH and *CH3CO has been confirmed as the rate-determining step for EOR103CO solely decomposes into *CO and *CH3We further carried out the density functional theory (DFT) calculation  to under pathway 1013. In athway 10, where t− shows preferential adsorption on Ni atoms, which is in good agreement with experimental observation− and Ni atom that carries significantly positive charge2628−  and the lowest desorption energy  for *OH, suggesting that the incorporation of Ni and P is favourable for both chemical absorption of OH− and desorption of free *OH, which thus facilitates the formation of CH3COOH , nickel (II) acetylacetonate (Ni(acac)2, 95%), trioctylphosphine (TOP) (90%), Nafion solution (5 wt%), Palladium on activated carbon  were purchased from Alfa Aesar. Oleylamine (OAm) (>70%) was purchased from Sigma Aldrich. NaOH, ethanol, cyclohexane, toluene, n-hexane and isopropanol were obtained from Beijing Chemical Reagent Company. Ketjen Black was obtained from Shanghai HESEN Electric Company. Milli-Q ultrapure water was utilized through all the experiments.Palladium (II) acetylacetonate (Pd(acac)α radiation (λ=1.5418 Å). The tested current and voltage were 40 mA and 40 kV, respectively. A 2θ ranging from 25° to 90° was covered in steps of 0.02° with a count time of 2 s. Elemental composition of the NPs was determined using an inductively coupled plasma mass spectrometer . The X-ray photoelectron spectrum (XPS) was performed on ESCALAB 250 .TEM images were obtained on a JEM-1200EX (JEOL) transmission electron microscope (TEM) at 100 kV. HRTEM images were recorded via a JEOL JEM-2100F transmission electron microscope operating at 200 kV. Powder XRD patterns were recorded on a Bruker AXS D8-Advanced X-ray diffractometer with Cu K38Ni49P13, Pd38Ni45P17 and Pd40Ni43P17 are similar. In a typical preparation of Pd38Ni45P17 NPs , the reaction was carried out under nitrogen flow and magnetic stirring. About 152.3 mg Pd(acac)2, 135.2 mg Ni(acac)2 and 20 ml OAm were added to a 50-ml three-necked round bottom flask. The mixture was then heated at 80 °C for 10 min to make sure all the reactants were totally dissolved. Then, 1.5 ml TOP was added to the blue transparent solution, thereafter the solution colour changed to green, and the temperature was increased to and kept at 120 °C for 30 min. The solution was then heated to 260 °C within 200 s under sufficient stirring and moderate nitrogen flow. At 230 °C, the solution exhibited a sharp change in colour and turned to black. Then, the temperature was maintained at 260 °C for 1 h. After the solution was cooled to room temperature naturally, the Pd38Ni45P17 NPs were collected by centrifugation at 13,000 r.p.m. and washed three times with an ethanol/cyclohexane mixture. The product was dispersed in 5 ml toluene for later use. Under the same condition, 122 mg Pd(acac)2 and 162.3 mg Ni(acac)2 were used to produce Pd31Ni53P16 NPs , and 183 mg Pd(acac)2 as well as 108.2 mg Ni(acac)2 were adopted to generate Pd47Ni36P17 NPs .The details for the preparation of the NPs are shown in 54Ni30P16 , Pd40Ni43P17  and Pd32Ni50P18 NPs  , Pd38Ni45P17  and Pd31Ni53P16  precursor NPs   were obtrsor NPs . In a ty−1. To make the same Pd loading on electrode (20 μg cm−2) as that of nanocatalysts, the concentration of commercial Pd/C catalyst ink is 4.8 mg ml−1.Seven milligrams of Ketjen Carbon were placed in centrifuge tubes before 3 ml n-hexane was added. The mixture was sonicated for 30 min to ensure the formation of good suspension. Then, 2 ml NPs n-hexane dispersion, containing approximately 7 mg NPs, was added into the Ketjen Carbon suspension. The mixture was then sonicated for 60 min to allow the NPs to be transferred onto the carbon support, which was indicated by the colourless supernatant. About 40 ml of ethanol was added and the mixture was sonicated for further 30 min and centrifuged at 12,000 r.p.m. for 15 min. The colourless supernatant was discarded. The process was repeated twice. The NPs/C product was dried at 60 °C for 12 h, and dispersed in the mixture of ultrapure water, isopropanol and Nafion solution (5 wt%) (v/v/v 3:3:0.2) by sonicated to yield a well-dispersed suspension as catalyst ink with a concentration of 2.4 mg mlE from E(SCE) by following the formula E(RHE)=E(SCE)+0.254+0.05916 × pH. The calibration was performed in the high purity hydrogen saturated electrolyte with a Pt foil as the working electrode. As shown in −1 in 1.0 M NaOH for several cycles to remove the residual ligands on catalyst surface. Afterwards, for the EOR measurement, the working electrodes were subject to CV scans between 0.18 and 1.38 V at 100 mV s−1 in 1.0 M NaOH and 1.0 M ethanol. The chronoamperometry measurements were conducted at 0.8 V in the solution of 1.0 M NaOH and 1.0 M ethanol. For CO-stripping tests, CO oxidation experiments were carried out in the solution of 1.0 M NaOH. Before the test, the solution was purged with nitrogen for 30 min and then was bubbled with CO gas (99.9%) for 15 min at 0.18 V to achieve the maximum coverage of CO at the Pd active centres. The residual CO in the solution was excluded by nitrogen for 30 min.CV measurements were carried out in a three-electrode cell using electrochemical workstation . Our experiments were performed with a saturated calomel electrode (SCE) electrode as the reference electrode. It was calibrated to Data supporting the findings of this study are available within this article and its How to cite this article: Chen, L. et al. Improved ethanol electrooxidation performance by shortening Pd–Ni active site distance in Pd–Ni–P nanocatalysts. Nat. Commun.8, 14136 doi: 10.1038/ncomms14136 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Method and Supplementary References