The immunosensor was able to recognize three different His-tagged variants of recombinant hemagglutinin from H5N1 viruses: H1 subunit (17–340 residues) of A/swan/Poland/305-135V08/2006, the long HA (17–530 residues) A/Bar-headed Goose/Qinghai/12/2005 and H1 subunit (1–345 residues) of A/Vietnam/1194/2004. The strongest response has been observed for the long variant with detection limit of 2.2 pg/mL and dynamic range from 4.0 to 20.0 pg/mL.This paper concerns the development of an immunosensor for detection of peptides derived from avian influenza hemagglutinin H5. Its preparation consists of successive gold electrode modification steps: (i) modification with 1,6-hexanedithiol and gold colloidal nanoparticles; (ii) immobilization of antibody-binding fragments (Fab') of anti-hemagglutinin H5 monoclonal antibodies Mab 6-9-1 via S-Au covalent bonds; and (iii) covering the remaining free space on the electrode surfaces with bovine serum albumin. The interactions between Fab' fragments and hemagglutinin (HA) variants have been explored with electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN) It is an enveloped RNA virus about 100 nm in diameter with an eight-segmented, single-stranded and negative-sense genome. Two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), are present on the surface and play important roles in infecting a host cell. Sixteen HA (H1–H16) and nine NA (N1–N9) subtypes of the influenza virus are found 3−/4− as an electroactive marker.Here, we present a sensitive immunosensor for the detection of peptides derived from avian influenza hemagglutinin H5 based on the recognition of the epitope localized in the N-terminus of HA by polypeptide Fab' from monoclonic Mab 6-9-1. Specific exposure of thiol groups by the Fab' fragment allows for random dense and precise saturation of the sensor surface with antibodies. This simplifies the preparation of electrode surfaces and sensing of the HA antigen composition. Specific interactions between oriented Fab' fragments immobilized on the gold electrode surface and three different His-tagged variants of recombinant hemagglutinin: H1 subunit (17–340 residues) of A/swan/Poland/305-135V08/2006 (Clade 2.2), H1 subunit (1–345 residues) of A/Vietnam/1194/2004 (Clade 1) and the truncated HA  A/Bar-headed Goose/Qinghai/12/2005 (Clade 2.2) were observed using EIS in the presence of /K4[Fe(CN)6] (0.5 mM each). In the CV potential were cycled from 0.6 to −0.2 V with scan rate 0.1 V/s. The EIS were recorded within the frequency range of 0.1 Hz to 10 kHz at formal potential of the redox couple [Fe(CN)6]3−/4− (0.17 V) with ac amplitude of 10 mV. Obtained spectra were fitted by the AutoLab software in order to obtain values of electron transfer resistance (iR). The electrode responses were expressed as: (iR−R0)/R0 where R0 means electron transfer resistance of fully modified electrode measured in pure PBS buffer before His-tagged recombinant proteins detection, iR means electron transfer resistance of fully modified electrode measured in PBS containing particular concentration of the detected proteins.All electrochemical measurements were performed at room temperature with an AutoLab potentiostat-galvanostat  using a three-electrode configuration. Working electrodes were polycrystalline gold discs 2 mm diameter . All potentials were measured versus an Ag/AgCl reference electrode and platinum wire was used as the counter electrode. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were performed in solution comprised of 0.1 M PBS pH 7.4 and K3.3.1.The process of immunosensor fabrication is shown in 3[Fe(CN)6]/K4[Fe(CN)6] as redox probe in PBS showed a couple of redox peaks with ΔEp = 75 mV  vs. imaginary (imZ) impedance (etR = 1359.3 kΩ). The chemisorptions of GCP on the 1,6-HDT layer decreased the electron transfer resistance to 112.4 kΩ  with different sensitivity. The highest concentration of antigen (20 pg/mL) caused a significant increase of the electron transfer resistance to 23.9 ± 2.0% for Qinghai, 19.4 ± 1.9% for HA/Nde and 10.3 ± 2.5% for Vietnam .S is the slope of the calibration curve [iR − R0)/R0c] pg/mL for Qinghai, 1.0%[(iR − R0)/R0c] pg/mL for HA/Nde and 0.6%[(iR − R0)/R0c] pg/mL for Vietnam. The immunosensor presented in this paper thus has good sensitivity. This is mainly because of the immobilization strategy used. The covalent bonds between Au atoms of GCP and SH group of Fab' fragments enable stable and appropriate orientation for antigen binding.Limits of detection (LOD) were calculated based on the standard deviation of the response and the slope of the calibration curve:on curve . Limits Parental monoclone Mab 6-9-1 is presumably directed toward a conformational epitope located within the H1 domain of the HA protein of H5N1 (A/swan/Poland/305-135V08/2006) virus. As expected, the sensor equipped with Fab' fragments of this monoclone efficiently recognizes the HA/Nde antigen representing H1 domain coded by homologous virus as well as Qinghai polypeptide coded by the virus from the same IV Clade 2.2.The immunosensor shows lower affinity to Vietnam antigen expressing the similar H1 region, however coded for by the strain belonging to the distant H5N1 virus Clade 1. When calculating sensitivity in molar antigen concentrations, the proposed immunosensor shows the highest affinity toward Qinghai polypeptide equipped besides the H1 domain with further fragments of HA. This additional region (between aa 341-530) does not carry epitopes recognized by the parental Mab 6-9-1. The observed rise in sensitivity suggests that the additional HA polypeptide fragment is implicated in exposition of the H1 domain, confirming the claim that Mab 6-9-1 recognizes conformational epitope. The proposed sensor equipped with Fab' fragments follows the characteristics of the parental monoclone in distinguishing the conformers of the same epitope.We are aware that final validation of the sensor requires testing its specificity against a collection of antigens from alien respiratory viruses. The characterisation of parental Mab 6-9-1 specificity is in progress  and this will lead to the requisite validation experiments.6-rSPI2 protein [6-rSPI2 protein improves the detection limit to 5 pg/mL [In our the previous paper we created an immunosensor based on gold electrodes modified with 1,6-HDT, GCP, whole IgG antibodies and BSA for detection of His protein  and the  protein  (Table 1 5 pg/mL  in compa 5 pg/mL , proving 5 pg/mL ,27,39,45 5 pg/mL ,41,46 (TIt is worth emphasizeing that the main advantages of the immunosensor proposed here are very small sample demand, good sensitivity and simple fabrication, with the possibility for miniaturization. The detection limits for nanoscale biosensors are mainly governed by analyte transport limitation towards sensing layers, not a signal transduction . The sen4.Concluding, a sensitive and selective impedimetric immunosensor for the detection of peptides derived from avian influenza hemagglutinin H5 using Fab' immobilized on a gold electrode surface via colloidal gold nanoparticles was developed. This device is able to recognize three different His-tagged fragments of HA: Qinghai, HA/Nde and Vietnam. The strongest response was observed for Qinghai, with a detection limit of 2.2 pg/mL and a dynamic range from 4.0 pg/mL to 20.0 pg/mL. The presented research shows that gold colloidal nanoparticles may be used for the creation of a very good underlayer for Fab' oriented immobilization. They allow fragments of antibodies to retain their activity and constitute a good electron conductive layer for electrochemical sensors. Considering its good selectivity and sensitivity in the pg/mL range, the proposed immunosensor was superior in comparison to others already reported, therefore, it could be recommended for the rapid, simple and direct electrochemical detection of avian influenza virus H5N1.


Spodoptera litura), an important agricultural pest, causes severe yield losses in many crops. In this study, we identified CYP9A40, a novel P450 gene of S. litura, and investigated its expression profile and potential role in detoxification of plant allelochemicals and insecticides. The cDNA contains an open reading frame encoding 529 amino acid residues. CYP9A40 transcripts were found to be accumulated during various development stages of S. litura and were highest in fifth and sixth instar larvae. CYP9A40 was mainly expressed in the midgut and fat body. Larval consumption of xenobiotics, namely plant allelochemicals (quercetin and cinnamic acid) and insecticides (deltamethrin and methoxyfenozide) induced accumulation of CYP9A40 transcripts in the midgut and fat body. Injection of dsCYP9A40 (silencing of CYP9A40 by RNA interference) significantly increased the susceptibility of S. litura larvae to the tested plant allelochemicals and insecticides. These results indicate that CYP9A40 expression in S. litura is related to consumption of xenobiotics and suggest that CYP9A40 is involved in detoxification of these compounds.Cytochrome P450 monooxygenases (P450s) of insects play crucial roles in the metabolism of endogenous and dietary compounds. Tobacco cutworm moth ( Cytochrome P450 monooxygenases (P450s or CYPs) represent a large enzyme family, with representatives in most organisms . P450s oSpodoptera litura Fabricius) is a well-known pest that causes severe yield losses of many important crops such as soybean, tomato, potato, cotton and groundnut [S. litura has become increasingly difficult all over the world, particularly in many Asian countries [S. litura are mainly attributed to its ability to adapt to a wide array of host plants and development of resistance to commonly applied insecticides [S. litura can tolerate in its diet considerable amounts of plant allelochemicals or pesticides [S. litura, while cinnamic acid is rarely encountered by S. litura larvae [S. litura larvae. Deltamethrin [S. litura control, deltamethrin and methoxyfenozide are mainly used as larvicidal or adulticidal agents [The polyphagous tobacco cutworm moth  is a powerful tool to silence target genes, e.g., genes required for insect development and insect-plant relationships . SilenciS. litura to tolerate plant allelochemicals and insecticides, we became interested in the role of P450 genes of this pest [P450 gene from S. litura, named CYP9A40. We investigated the expression of CYP9A40 during different developmental stages and in different tissues. We also explored the function of CYP9A40 by using RNAi-silenced larvae that were fed with diets containing plant allelochemicals or insecticides. The results suggest a role of CYP9A40 in detoxification of plant allelochemicals and insecticides.To better understand the ability of his pest . In thisCYP9A40, a cytochrome P450 gene of S. litura, was cloned. The full-length 1639-bp cDNA sequence of CYP9A40 contains a 38 bp 5′-untranslated region (5′-UTR), an open reading frame (ORF) of 1590 bp, and an 11-bp 3′-untranslated region (3′-UTR). The sequence was deposed at the GeneBank database (accession number: KR065418). The ORF encodes a predicted protein of 529 amino acids. CYP9A40 has a theoretical pI value of 8.57 and a predicted molecular mass of 61.48 kDa. CYP9A40 contains the signature motif of P450s and shares highest amino acid sequence identity with members of the CYP9A subfamily [CYP9A40 with three CYP9A subfamily members from Helicoverpa  [ubfamily ,13. By aicoverpa , it was s (SRSs)  were alsHeliothis virescens. CYP9A40 and CYP9A1 have an amino acid identity of 70.7%. The amino acid identities of CYP9A40 with CYP9A3 of Helicoverpa armigera and CYP9A58 of Spodoptera frugiperda are 69.4% and 56.4%, respectively , fifth (60-fold higher than in eggs) and sixth (65-fold higher than in eggs) instar larvae. Expression of CYP9A40 was significantly higher in fifth and sixth instar larvae than in the other developmental stages. Transcripts of CYP9A40 were also elevated in pupae (21-fold higher than in eggs).qRT-PCRs were conducted to determine the expression pattern of . litura A. TranscCYP9A40 in different tissues. RNAs were extracted from cuticle, brain, midgut, fat body, ovary and hemolymph dissected from sixth instar larvae , midgut (44-fold higher than in hemolymph) and ovary (16-fold higher than in hemolymph). In contrast, gene expression of CYP9A40 was found to be relatively low in the cuticle, brain and hemolymph.We further investigated expression levels of r larvae B. qRT-PCCYP9A40 transcripts, qRT-PCR was performed using 2-day old fifth instar larvae fed for 48 h with artificial diet supplemented with quercetin, cinnamic acid, deltamethrin and methoxyfenozide. Compared to control larvae , CYP9A40 expression in the midgut significantly increased in response to uptake of quercetin (6.5-fold), cinnamic acid (5.3-fold), deltamethrin (6.2-fold) and methoxyfenozide (3.9-fold)   . A similenozide) . Hence, CYP9A40 expression in fifth instar larvae was silenced by an RNAi approach. Microinjection was performed with dsCYP9A40 and dsGFP served as a control. Expression analysis of CYP9A40 by qRT-PCR was conducted 24 h after delivery of dsRNA. Compared to control larvae, larvae injected with dsCYP9A40 showed significantly lower levels of CYP9A40 transcripts (decrease to 24.2% in the midgut and to 26.3% in the fat body). These results indicate considerable silencing of CYP9A40 by RNAi  increased larval mortality caused by cinnamic acid (from 51.7% to 70.8%) and quercetin (from 45.8% to 66.7%). Similar changes were also observed for deltamethrin (increase from 55.8% to 79.1%) and methoxyfenozide (from 61.7% to 85.8%) (We further used the o 85.8%) B. These S-transferase and carboxylesterases [S. litura encounters large quantities of plant allelochemicals and insecticides. In this study, we cloned and characterized the P450 gene CYP9A40 of S. litura. The amino acid sequence of CYP9A40 contains various motifs conserved in P450s including predicted SRSs , when sixth-instar larvae of H. armigera were exposed to the P450 gene inducer phenobarbital [Herbivorous insects have evolved a variety of mechanisms to adapt to allelochemicals of host plants and insecticides. Adaptation includes production of detoxifying enzymes such as P450s, glutathione sterases ,15. P450ted SRSs . SRS1, Sectively . CYP9A1 iodicarb . CYP9A58fenozide . Expressbarbital .CYP9A40 in S. litura, we determined expression levels of CYP9A40 at different development stages and in different tissues. Highest transcript levels were found in fourth to sixth instar larvae, particularly in the midgut and fat body  [Expression pattern of inducible genes can provide useful information on their biological functions ,17. To cfat body . The midfat body . The expcuticle) .Bombyx mori), for example, the activity of P450s in the midgut was 2.3-fold stimulated after feeding larvae with diet supplemented with quercetin [H. armigera exposed to fenvalerate (a type II synthetic pyrethroid) for 14 generations, transcripts of CYP9A12, CYP9A14, and CYP6B7 showed strongly induced levels in the fat body of larvae [CYP9A12 and CYP9A14 in yeast suggests that these P450s of H. armigera can play role in detoxification of insecticides such as esfenvalerate (the (S)-enantiomer of fenvalerate) [CYP6AE11, another P450 gene of H. armigera, was found to be strongly expressed in pyrethroid resistant strains and it was suggested that CYP6AE11 also plays a role in detoxification of xenobiotics [CYP6B6 gene were found to be strongly accumulated in the midgut and fat body of H. armigera and expression of this gene was stimulated in a feeding experiment with plant allelochemicals (2-tridecanone and quercetin). Transcript levels of this gene correlated with the concentration of applied 2-tridecanone but not with the quercetin concentration [Helicoverpa zea, expression of four CYP6B genes  was increased in response to plant allelochemicals, namely coumarin, quercetin, xanthotoxin, and flavone [H. zea have been demonstrated to metabolize insecticides  as well as plants allelochemicals such as xanthotoxin [S. litura, expression levels of CYP6B58 in the midgut were slightly enhanced by uptake of xanthotoxin (1.7-fold) or coumarin (1.5-fold) [S. litura, namely CYP6B48 and CYP6AB14, likely metabolize plant allelochemicals such as cinnamic acid, quercetin, coumarin, xanthotoxin and flavone [CYP9A40, indicating up-regulated gene expression by a broad range of xenobiotics  . Expressobiotics . Moreoventration . In Heli flavone . CYP6B8 thotoxin . In S. lobiotics . The obsP450 genes in detoxification of plant allelochemicals and insecticides [S. litura larvae resulted in a noticeable reduction of CYP9A40 transcripts in the midgut and fat body. Remarkably, injection of dsCYP9A40 followed by feeding with quercetin, cinnamic acid, deltamethrin or methoxyfenozide significantly increased mortality rates of the instar larvae  also resulted in increased mortality rates of S. litura larvae [P450 genes in other insects. In H. armigera for example, silencing of CYP6AE14 by RNAi significantly reduced the growth of larvae that were fed with cotton (Gossypium hirsutum cv. R15) [CYP321E1 in the moth Plutella xylostella resulted in increased mortality of fourth-instar larvae exposed to the insecticide chlorantraniliprole [CYP9AQ2 expression by RNAi led to an increased mortality of third-instar nymphs of Locusta migratoria that were exposed to the insecticide deltamethrin [S. litura cells should be analyzed in further studies.RNAi is a powerful method to assess the roles of cticides ,27,28. Ir larvae B. These cv. R15) . Likewisiliprole . FurtherCYP9A40, a novel P450 gene of the moth S. litura. CYP940 expression varied with developmental stages and tissues. It was highest in the midgut and fat body of sixth instars. Uptake of plant allelochemicals and insecticides stimulated expression of CYP9A40 in larvae. Silencing of CYP9A40 in larvae led to increased larval susceptibility to these compounds, suggesting an important role of CYP9A40 in detoxification of xenobiotics. Future work is required to characterize the CYP9A40 protein and to examine synergistic effects with other P450s of S. litura.In summary, we identified and characterized S. litura were provided by the Insectarium of the Institute of Entomology, Sun Yat-sen University  and maintained in an insectary without exposure to any insecticides and chemicals at South China Agricultural University . Larvae of S. litura were feed on artificial diet [Larvae of ial diet ,19 and whttp://blast.ncbi.nlm.nih.gov) using expressed sequence tag (EST) sequences derived from RNA isolated from the midgut of sixth instar larvae of S. litura [CYP9A40 gene (coding sequence and untranslated regions). The amplification conditions were as follows: 95 °C for 2 min, 35 cycles  and a final extension step of 72 °C for 10 min. The PCR product was ligated into the pGEM-T Easy Vector  and sequenced using an ABI 377 capillary automated sequencer. The open reading frame (ORF) of CYP9A40 was obtained by the ORF Finder program at the NCBI homepage (Available online: http://www.ncbi.nlm.nih.gov/gorf/gorf.html).CYP9A subfamily member-like nucleotide sequences were identified by a Blast similarity search  according to the manufacture’s instructions. The quality and concentration of RNA were examined by agarose gel electrophoresis and spectrophotometer analysis. Extracted RNAs were treated with DNase I (Invitrogen) and then reverse transcribed using the ThermoScript™ RT-PCR System kit  according to the instructions of the provider.S. litura, CYP9A1 from H. virescens (tobacco budworm), CYP9A4 and CYP9A5 from Manduca sexta (goliath worm), CYP9A3, CYP9A12, CYP9A14, CYP9A17 and CYP9A18 from H. armigera (cotton bollworm), CYP9A19, CYP9A21 and CYP9A22 from B. mori (silkworm) and CYP9A58 from S. frugiperda . The phylogenetic tree was built using Mega 4.0 software  [H. virescens and CYP9A17 of H. armigera as described by Zhou et al. [Phylogenetic analysis was performed for 12 amino acid sequences, which were considered to be representative for lepidopteran P450s involved in metabolism of xenobiotics: CYP9A40 from AZ, USA)  and the AZ, USA) . SubstraCYP9A40 were determined by quantitative real-time RT-PCR (qRT-PCR). The gene-specific primers CYP9A40F (5′-TACAGGCTTCTACCATCCG-3′) and CYP9A40R (5′-TGGCGTCATACTCCCTAAT-3′) were used for detection of CYP9A40 transcripts. The β-actin gene was used as an internal standard gene to normalize the expression levels among different samples. The primers of the β-actin gene  have been used before [CYP9A40 were determined by using the 2Ct−ΔΔ method [Relative expression levels of d before . Real-tit method .CYP9A40 expression pattern at different development stages of S. litura, RNA was extracted from eggs (30 eggs per RNA extraction), first to sixth instar larvae at day 2 (4 larvae per RNA extraction) and pupae at day 2 (3 pupae per RNA extraction). For analysis of the CYP9A40 expression pattern in different tissue types, RNA was extracted from cuticle, brain midgut, fat body, ovary and hemolymph from fifth instar larvae. RNA isolation and qRT-PCR were done as described above. Three independent RNA extractions were performed for each analyzed biological material.For analyses of the S. litura were fed for 48 h on artificial medium [w/w) quercetin , 1% (w/w) cinnamic acid , 0.05% (w/w) deltamethrin  and 0.03% (w/w) methoxyfenozide . After 48 h, both midgut and fat body were dissected from surviving larvae. The material was then immediately frozen in liquid nitrogen and kept at −80 °C for RNA extraction and further CYP9A40 expression analysis. Twenty individuals were used for each treatment. Three independent RNA extractions  were performed for all treatments.Newly molted fifth instar larvae of l medium  containiCYP9A40 expression in fifth instar larvae. The CYP9A40 cDNA templates for in vitro transcription reactions were prepared by PCR using cDNA (derived from RNA of 4 fifth instar larvae) as template. Two pairs of primers (dsCYP9A40 F (5′-AATACGACTCACTATAGGGTACAGGCTTCTACCATCCG-3′) and CYP9A40 R (5′-TGGCGTCATACTCCCTAAT-3′) (nucleotide position 1069–1087 in the ORF), CYP9A40 F (5′-TACAGGCTTCTACCATCCG-3′) (nucleotide position 676–694 in the ORF) and dsCYP9A40 R (5′-AATACGACTCACTATAGGGTGGCGTCATACTCCCTAAT-3′)) were used to synthesize dsRNA for CYP9A40 (676–1087 bp of ORF), which contained the T7 promoter region in both sense and antisense strands. The primers were designed based on the sequence of CYP9A40 and the amplification conditions were 94 °C for 5 min, 35 cycles  and a final extension step (72 °C for 10 min). As a control, dsGFP  was synthesized by the same PCR method using the following primer pairs: T7GFPdsRNA F (5′-AATACGACTCACTATAGGGAAGGGCGAGGAGCTGTTCACCG-3′) and GFPdsRNA R (5′-CAGCAGGACCATGTGATCGCGC-3′); GFPdsRNA F (5′-AAGGGCGAGGAGCTGTTCACCG-3′) and T7GFPdsRNA R (5′-AATACGACTCACTATAGGGCAGCAGGACCATGTGATCGCGC-3′) [CYP9A40 (dsCYP9A40) and GFP (dsGFP) were synthesized using a T7 RiboMAX™ Express RNAi System  according to the manufacturer’s instructions. The final concentrations of dsCYP9A40 and dsGFP were adjusted to 1.5 µg·µL−1 by DEPC-treated (RNase-free) water and kept at −80 °C. For all dsRNA injection experiments, fifth instar (day 2) larvae were used and 2 µL (3.0 µg) of dsRNA were injected into the side of the thorax of each larva using a manual microinjector . The injection points were sealed immediately with paraffin [S. litura were kept at 25 ± 2 °C, 70% ± 5% relative humidity and a 16-h light/8-h dark photoperiod.An RNAi approach was used to silence GCGC-3′) ,35. PCR paraffin . After iCYP9A40 were analyzed 24 h after the injection of dsCYP9A40 or dsGFP. RNA extraction and expression analysis of CYP9A40 by qRT-PCR were performed as described above. In total, 32 fifth instar (day 2) larvae were used for the CYP9A40 expression analysis. Each extracted RNA sample corresponded to four larvae .Expression levels of . After 24 h, mortality rates (ratio of dead larvae to the sum of tested larvae) were recorded for each subgroup (n = 4). The bioassays were performed in triplicate.For bioassays with plant allelochemicals and insecticides, microinjections of dsCYP9A40 or dsGFP were done for totally 240 fifth instar (day 2) larvae, which were randomly assigned into two groups before microinjection (120 each). The larvae were injected with 2 µL of dsCYP9A40 or dsGFP as described above. After dsRNA delivery, the larvae from each group were assigned into four subgroups (30 each), and individuals in each subgroup were kept on artificial diets containing plant allelochemicals or insecticides at concentrations described abovet-test was used to analyze data from the RNAi and feeding experiments with different xenobiotics. Statistical differences were considered significant at p < 0.05.Data were expressed as means ± standard error (SE). Statistical analysis was carried out with the SPSS 10.0 software package . One-way ANOVA followed by the Duncan’s multiple range test was employed to analyze differences among different tissues and development stages. The Students’s


HLA genes and T1D in a Chinese case-control cohort recruited from multiple centers consisting of 364 patients with T1D and 719 unrelated healthy children. We genotyped 55 single nucleotide polymorphisms (SNP) markers located in 16 non-HLA genes  using SNaPshot multiple single-base extension methods. After multivariate analysis and correction for multiple comparisons, we identified the SNP rs2292239 in ERBB3 gene were significantly associated with T1D. The frequency of the major G allele was significantly decreased in patients with T1D , and the minor allele T was associated with an increased risk of T1D . Our haplotype analysis confirmed that rs2292239 was the primary T1D association locus in our current investigation. These results indicated that the ERBB3-rs2292239 was the primary T1D association locus among the investigated 55 SNPs in 16 non-HLA genes in Chinese Han population.Type 1 diabetes (T1D) is an autoimmune disease that has strong contribution of genetic factors to its etiology. We aimed to assess the genetic association between non- •HLA genes.A large scale case-control genetic association study on type 1 diabetes in Chinese investigating on-•ERBB3 gene conferred the primary non-HLA association in Chinese type 1 diabetes.rs2292239 in the •Markers in the common candidate genes, such as PTPN22, CTLA4, IL2RA, and INS, were not significantly associated with T1D in our Chinese cohort. It was PTPN22, CTLA4, IL2RA, INS, CD69, ERBB3, CYP27B1, PTPN2, UBASH3A and IL2RB (SUMO4) (CD274), B7-H3 (CD276) and B7-H4 (VTCN1) (DHCR7 and VDR) genes (Recent genome-wide association studies (GWAS) revealed many T1D association loci located in genes related to T1D autoimmunity, including nd IL2RB . In addi (SUMO4) ; members (VTCN1) ; genes iR) genes  were alsPTPN22 and revealed the heterogeneity in PTPN22 association between Chinese Han and Caucasians (HLA genes by genotyping 55 single nucleotide polymorphisms (SNPs) in VTCN1, PTPN22, CTLA4, SUMO4, CD274, IL2RA, INS, DHCR7, ERBB3, VDR, CYP27B1, CD69, CD276, PTPN2, UBASH3A and IL2RB genes.Chinese Han population is among the East Asians with the lowest incidence of T1D around the world . Similarucasians . Neverthucasians . Therefo22.1We recruited a case-control study cohort involving 364 patients with T1D and 719 unrelated healthy children. These subjects of Chinese Han origin were recruited in six medical centers across China as previously described in details . The stu2.22 > 0.8. We performed genotyping of those SNPs using genomic DNA extracted from 500 μl peripheral blood of the subjects . The genotyping was carried out by SNaPshot multiple single-base extension (SBE) reaction using purified PCR products amplified from genomic regions containing target SNPs as previously described (We studied 55 SNPs that are mapped to genes previously implicated in the pathogenesis of T1D (described above in the introduction section and listed in escribed . Genotyp2.3www.r-project.org). Deviation from the Hardy-Weinberg equilibrium (HWE) was first evaluated using Pearson's χ2-test. The allelic association between each SNP and T1D were tested using logistic regression. P values < 0.05 after Bonferroni correction were considered statistical significant. We conducted haplotype analysis using Haploview (version 4.1) with 10,000 permutations; a P-value < 0.05 after Bonferroni correction was considered significant. Odds ratios (OR) and 95% confidence intervals (CI) of each haplotype were calculated using Fisher's exact test by comparing the haplotype frequencies predicted by Haploview.We conducted data analysis using R . However these associations were no longer significant after Bonferroni correction . Our results showed that the haplotypes in INS, PTPN2 and UBASH3A genes were not associated with T1D  were significantly associated with T1D. After Bonferroni correction, the G-G-A haplotype were significantly less frequent in patients with T1D than in controls  and the T-G-A haplotype was positively associated with T1D .To investigate whether there was combinational effect of SNPs that further affected the association between gene and T1D, we conducted haplotype analysis using genotyping data of the above identified four genes  and 55 SNPs in 16 non-ERBB3-rs2292239 and T1D has been identified in large scaled studies in many populations, including Caucasians  (ERBB3 was one of the loci with the most significant T1D association among non-HLA genes (ERBB3 gene encodes erb-b2 receptor tyrosine kinase 3 (ErbB3), a member of the epidermal growth factor receptor (EGFR) family  , Japanes OR 1.3)  and Paki OR 1.3) . SimilarLA genes . The ERB) family . Dislike) family , which t) family . However studies . Althoug11171747 . WhetherERBB3-rs2292239 however not in INS or IL2A  in the East Asian populations therefore studies results are more likely to be influenced by statistical stringency and other variations across study protocols. Furthermore, the weak association between non-HLA susceptible genes made the inter-ethnic association easily influenced by variations of allele distributions across populations , Chinese National Twelfth Five Year Key Science and Technology Plan (2012BAI09B00) and National Natural Science Foundation of China (NSFC-81500599).This study received financial supported from the The authors declare that there is no conflict of interest related to this article.


Host-adaptive strategies, such as the E627K substitution in the PB2 protein, are critical for replication ofavian influenza A viruses in mammalian hosts. Here we show that mutationPB2-K526R is present in somehuman H7N9 influenza isolates, in nearly 80% of H5N1 human isolates from Indonesiaand, in conjunction with E627K, in almost all seasonal H3N2 viruses since 1970.Polymerase complexes containing PB2-526R derived from H7N9, H5N1 or H3N2 viruses exhibit increasedpolymerase activity. PB2-526R alsoenhances viral transcription and replication in cells. In comparison with virusescarrying 627K, H7N9 viruses carrying both 526R and 627K replicate more efficientlyin mammalian (but not avian) cells and in mouse lung tissues, and cause greater bodyweight loss and mortality in infected mice. PB2-K526R interacts with nuclear export protein and our resultssuggest that it contributes to enhance replication for certain influenza virussubtypes, particularly in combination with 627K. Mutations in the viral polymerase, such as PB2-E627K, contribute toadaptation of avian influenza strains to mammalian hosts. Here the authors show thatanother mutation, PB2-K526R, is found in seasonal H3N2 and avian-origin human influenzaisolates, and facilitates replication of these viruses in mammals. Adaptation of viral polymerase is critical for efficient virus replicationin a new host following cross-species transmission13578http://www.who.int/influenza/human_animal_interface/EN_GIP_20140124CumulativeNumberH5N1cases.pdf).However, the majority (80%) of the H5N1 isolates from human cases in Indonesia do notharbour the PB2 627K or 701N markers.Apart from the markers described above, influenza viruses probably utilizeuncharacterized adaptive substitutions in the PB2 polymerase subunit to support virus replication in human cells.In the spring of 2013, a novel avian H7N9 virus emerged that crossed the species barrierto infect humans in eastern China19192026While about 50% of H5N1 human isolates contain known PB2 adaptation markers, mainly E627K with some D701N and a fewinstances of 591K, as described above, the other half of human infections are notassociated with known adaptation markers1617This study analyzes influenza sequences available from public databases and also thosearising from our analysis of H7N9 isolates from human cases to identify new adaptationmarker(s) in the PB2 gene. VariousPB2 adaptation markers areidentified among the H7N9 human isolates, and a K526R substitution associated with otherpreviously defined adaptive markers is detected in some human isolates. Furthercharacterization of the PB2-526Rgenotype reveals that H7N9 virus carrying this marker replicates to a significantlyhigher titre in mammalian cells, and also in mice. Furthermore, we show that nearly 80%of H5N1 human isolates from Indonesia and almost all seasonal H3N2 influenza A virusesisolated since 1970 contain PB2-526R.Reverse mutation of 526R to 526K attenuates polymerase activity in H3N2 andIndonesia-subclade H5N1 viruses. Therefore, in addition to 627K, 701N, 590S/591R and591K, 526R is another marker of mammalian adaption by avian influenza viruses, possiblyfunctioning through interaction between viral PB2 and nuclear exportprotein (NEP)during virus replication.Examination of the sequences of H7N9 human isolates has revealed a variety ofdifferent mammalian adaptation markers present in PB2 genes19202919292031The influenza A virus viral polymerase complex, which is composed ofPB1, PB2 and PA subunits, is used to replicate andtranscribe the viral genome in the cell nucleus. We tested whether the presenceof 526R, either alone or in combination with other adaptation markers, alteredthe ribonucleoprotein (RNP) complex polymerase activity of H7N9 virus. We firstcompared polymerase activity using a minigenome assay, testing all PB2 genotypes identified from H7N9viruses for which sequence data had been publicly released or obtained from ouranalyses . As expe33The seasonal H3N2 virus has been circulating continuously in humans since 1968,and is believed to have gained most of its adaptations through interaction withits human host. Since the 526R genotype was found among H3N2 viruses from around1970 onwards and has become prevalent since then, we tested whether gaining 526Renhanced virus replication. Introduction of 526R into the PB2 of an H3N2 strain pre-dating thischange (A/Hong Kong/1/68) significantly increased polymerase activity, which isconsistent with the observations for H7N9 PB2 . ConsistTo further understand whether the altered RNP polymerase activity and viralgrowth associated with 526R versions of PB2 is due to differences in the efficiency of virus genometranscription and replication, we examined levels of viral mRNA and vRNA in A549cells infected with viruses containing different versions of PB2 at 6 h post infection.Quantitative reverse transcription-PCR (RT-PCR) analysis with mRNA- orvRNA-specific primers showed that A549 cells infected with viruses carryingeither 627K, 701N or 526R PB2have significantly higher levels of viralmatrix protein (M1) and nucleoprotein (NP) mRNA and vRNA, as compared with cells infected with avirus containing avian PB2, suggestTo further characterize the phenotype of viruses carrying 526R PB2, reverse genetic (RG) versions ofviruses from human isolates carrying the 627K, 701N, 526R/627K, 526R/701N and solely526R PB2 genotypes wereconstructed in the A/Zhejiang/DTID-ZJU01/2013 backbone. Comparison of the growthkinetics of these viruses with a similarly constructed RG version of an avian H7N9strain (A/Chicken/Zhejiang/DTID-ZJU01/2013) revealed that viruses carrying any ofthe adaptation markers, including the novel 526R, replicated to a 1–2 loghigher titre than the avian H7N9 virus in Madin-Darby canine kidney (MDCK) cells. Our res50) of H7N9 viruses containing different genotypes ofPB2. Our results showedthat inclusion of K526R, alone or coupled with 627K or 701N, increased mortalityin the infected mice , based on pre-experimental data obtained using either 627K or 701NPB2 genotype viruses)dose of one of the different versions of H7N9 virus and were observed, andchanges in body weight recorded over the following 14 days. Viruses carrying526R or 627K substitutions caused moderate body weight loss  plus NP. Characterization of host adaptationmarkers among human isolates is important for recognizing potential forcross-species transmission in avian influenza A viruses. Adaptation of avianinfluenza A virus polymerases in mammals has been extensively studied and the mostwell-characterized adaptation markers are located in the PB2 polymerase subunit317Human infections with H7N9 virus emerged in China in March 2013 refs , 20, 31.4445464726The 526R PB2 mutation is rarelyfound among avian isolates and it was logical to presume that it may be anuncharacterized marker for human adaptation. Importantly, we found that the H3N2virus has also gained the PB2-K526R substitution, in addition to 627K; this occurred around1970, based on available sequence data, and this genotype has become prevalent inhumans since then ; Fig. 1.Protein structure data for the C-terminal domain of PB2, which contains the 627 and 701residues, suggests that either 627K or 701N may enhance PB2 exposure to allow better interactionwith elements of the host machinery such as importins, which facilitate nuclearimport during virus infection50Further studies are necessary to determine whether viruses that have gained dual526R/627K or 526R/701N substitutions may transmit more efficiently in humans, or inanimal models. Full evaluation of the transmission and replication abilities of H7N9and other avian influenza A viruses, such as H5N1-(Indonesia), bearing K526RPB2 will provide valuableinformation for understanding the adaptation process and establish the importance ofthis new marker in surveillance of zoonotic influenza A viruses.All influenza viruses used in this study were rescued using the RGs technique toensure the absence of contamination from other microbes. Viruses were propagatedin embryonated chicken eggs (9–11 days old) for 3 days (P1), usingthe standard protocol approved by the Committee on the Use of Live Animals inTeaching & Research (CULATR) of the University of Hong Kong.AGU02226 to AGU02234, KJ633805, andKJ473719 to KJ473726). Eight segments from the A/Zhejiang/DTID-ZJU01/2013and A/Chicken/Zhejiang/DTID-ZJU01/2013 strains were cloned into a pHW2000plasmid system3353http://gisaid.org) were analysed forprevalence of the K526R substitution.The H7N9 viruses from laboratory-confirmed human cases have been characterized inour previous studies20Renilla luciferase reporter plasmid (pRL_TK)construct (10 ng), then co-transfected into HEK293T cells andincubated at 37 °C or 33 °C, suppliedwith CO2. For luciferase reporter assays in DF-1 cells, a luciferasereporter containing a chicken RNA polymerase I (Pol I)-driven promoter wasconstructed based on the sequence previously describedLuciferase activity-based minigenome reporter assays were performed as describedpreviously−1 ofL-(tosylamido-2-phenyl) ethylchloromethyl ketone (TPCK)-treated trypsin. Infected cells were further incubatedat 37 °C, supplied with 5% CO2. Culturesupernatants were collected at different time points post infection and viraltitres determined by the plaque assay in MDCK cells. Confluent DF-1 cells wereinfected with RG viruses at an MOI of 0.01. The viral inoculums were removedafter 1 hours’ adsorption at 39 °C, then cellswere washed with MEM and overlaid with MEM containing0.5 μg ml−1 ofTPCK–trypsin.Infected cells were further incubated at 39 °C, suppliedwith 5% CO2. Culture supernatants were collected at different timepoints post infection and viral titres determined by the standard 50% tissueculture infective dose assay in MDCK cells.Confluent MDCK cells were infected with RG viruses at a multiplicity of infection(MOI) of 0.001. The viral inoculums were removed after 1 h ofadsorption at 37 °C and replaced with minimal essentialmedia (MEM) containing1 μg ml50 determination,groups of four or six mice were anesthetized with isoflurane , andintranasally inoculated with 25 μl of 10-fold serialdilutions of RG virus in PBS, the dose range being104–106 PFU. Body weightand survival were monitored daily for 14 days after infection. TheMLD50 of RG viruses were calculated by the method of Reed andMuench4 PFU (25 μl) of H7N9 RGviruses. Animals were observed daily for mortality and body weight was measuredfor up to 14 days after infection. At 72 h post infection, three micefrom each of the groups infected with virus containing 526R/701N, 701N,526R/627K or 627K PB2,avian-PB2,avian-PB2-526R or with RGavian H7N9 virus, or mock-infected with PBS were euthanized and lung tissueswere collected from each mouse for virus titration and histochemical staining.The protocols for animal experiments and the use of embryonated chicken eggs(9–11 days old) were approved by the CULATR, Li Ka Shing Faculty ofMedicine, the University of Hong Kong (CULATR 3064-13). The CULATR follows theHong Kong legislation and standards/guidelines recommended by the Associationfor Assessment and Accreditation of Laboratory Animal Care International.Female BALB/c mice, aged 4–6 weeks, were obtained from the LaboratoryAnimal Unit, the University of Hong Kong. For MLDTo test the specificity of the qPCR assay for viral mRNA, an NP-expressing plasmid was transfectedinto HEK293T cells for 48 h. Total RNA was extracted using the RNAisoreagent (Takara). Plasmid DNA present in the total RNA was eliminated bydeoxyribonuclease (DNase) treatment (Ambion), in accordance with the usermanual. Purified viral particles were used to test the specificity of the qPCRassay for vRNA. Total vRNA was again extracted using RNAiso. Complementary DNAwas synthesized using oligo dT, uni-12 or uni-13 primers, and the copy number ofNP was then quantified byqPCR using NP-specificprimers.−1) . For detection of vRNA and mRNA,uni-12 primer and oligo dT primer, respectively, were used in the RT reaction.Expression of these two RNA species of the NP and M1genes was quantified using SYBR Premix Ex Taq reagent (Takara) in an ABI 7500real-time PCR machine (Applied Biosystems Inc.). For the detection of theM1 gene, M1-72s(5′-cgcacagagacttgaggatg-3′) andM1-270as(5′-tgggtctccattcccattta-3′)primers were used. For the detection of NP, NP-545s(5′-cagtgaaggggatagggaca-3′) andNP-746as(5′-ccaggatttctgctctctcg-3′)primers were used. β-actin expression was detected for each sample andused for normalization of gene expression between different samples. Theamplification programme was as follows: 95 °C for30 s, followed by 40 cycles of 95 °C for5 s, 60 °C for 30 s. The specificityof the assay was confirmed by melting-curve analysis at the end of theamplification programme  ref..Mutation at the PB2 526position was performed using the QuikChange site-directed mutagenesis kit(Agilent) and RG H3N2 wild-type and PB2 mutant viruses were rescued, as previouslydescribedTo study the interaction between NEP and PB2, HEK293T cells were transfected with green fluorescentprotein (GFP)-tagged NEPexpression plasmid and FLAG-tagged PB2 expression plasmid. After 48 h, cells werelysed in cell lysis buffer . Cell debriswas cleared by centrifugation, and 1 μl of GFP antibody(Invitrogen) was added to the cell lysate, which was then incubated for3 h at 4 °C. Thirty microliters of protein Asepharose beads  were then added to the lysate, followed byincubation for 1 h at 4 °C. The beads were washedwith cell lysis buffer and finally boiled in SDS buffer at95 °C. Proteins were resolved withSDS–polyacrylamide gel electrophoresis and detected by western blotusing anti-GFP  (Invitrogen), anti-FLAG  andanti-β-actin  (Sigma) antibodies. To study theinteraction between NP andPB2 in RNP complexes, pCXplasmids expressing PB1,PA, NP and FLAG-tagged PB2 were co-transfected into HEK293Tcells. The immunoprecipitation steps were performed as described above, exceptthat a laboratory-made polyclonal NP antibody  was used.Statistical analysis was carried out using GraphPad Prism V5 software (GraphPadSoftware Inc.).W.S. and P.W. performed the research, analysed and interpreted the results and wrotethe manuscript. B.W.-Y.M., S.-Y.L., X.H., W.-L.W., M.Z., X.W., S.Y. and Y.C.performed animal experiments, virus isolation and analysed data. L.L. and K.-Y.Y.co-directed the study. H.C. directed the study, analysed and interpreted results andwrote the manuscript.How to cite this article: Song, W. et al. The K526R substitution inviral protein PB2 enhances theeffects of E627K on influenza virus replication. Nat. Commun. 5:5509 doi:10.1038/ncomms6509 (2014).Accession codes: Nucleotide sequences for the H7N9 and H3N2 viruses used inthis study have been deposited in the NCBI GenBank database with accession codesAGU02226 to AGU02234, KJ633805, andKJ473719 to KJ473726.Supplementary Figures 1-6


P<0.05) at 24, 48 and 72hpi, especially F-actin-capping protein subunit alpha-1 (CAPZA1), Ornithine aminotransferase (OAT), Poly(rC)-binding protein 1 (PCBP1), Eukaryotic translation initiation factor 5A-1 (EIF5A) and Platelet-activating factor acetylhydrolaseⅠb subunit beta (PAFAH1B2) were validated by western-blot analysis. The functional analysis revealed that the differential proteins in A549 cells involved in regulating cytopathic effect. Among them, the down-regulation of CAPZA1, OAT, PCBP1, EIF5A are related to the death of cells infected by H7N9 influenza virus. This is the first time show that the down-regulation of PAFAH1B2 is related to the later clinical symptoms of patients infected by H7N9 influenza virus. These findings may improve our understanding of pathogenic mechanism of H7N9 influenza virus in proteomics.A newly emerged H7N9 influenza virus poses high risk to human beings. However, the pathogenic mechanism of the virus remains unclear. The temporal response of primary human alveolar adenocarcinoma epithelial cells (A549) infected with H7N9 influenza virus and H1N1 influenza A virus  were evaluated using the proteomics approaches (2D-DIGE combined with MALDI-TOF-MS/MS) at 24, 48 and 72 hours post of the infection (hpi). There were 11, 12 and 33 proteins with significant different expressions ( Since March 2013, A novel H7N9 influenza virus first crossed the species barrier and infected humans in East China, causing disease with unusually high morbidity and mortality. It is a reassortant virus which consists of the gene fragments from earlier H7N9, H7N3 and H9N2 viruses . The patIn recent years, many new discoveries have been made concerning H7N9 infection. Sakai et al  demonstrAll procedures performed in studies were in accordance with the ethical standards of the institutional and/or national research committee and with the Helsinki declaration and were approved by the Medical Ethics Committee of Shenzhen Center for Disease Control and Prevention (SZCDC), Shenzhen, P. R. China.A549 cell line, H7N9 and H1N1pdm09 influenza virus were obtained from Shenzhen Center for Disease Control and Prevention (SZCDC). All the reagents for DIGE were purchased from GE Healthcare . DL-Dithiothreitol (DTT), Iodoacetamide (IAA), CHAPS were purchased from Sigma–Aldrich . The antibodies against CAPZA1, OAT, PCBP1, EIF5A, PAFAH1B2 were purchased from Abcam . The antibody against β-action was purchased from Santa Cruz . The secondary antibodies of goat anti-mouse and goat anti-rabbit were purchased from Thermo Fisher . The restriction enzyme (BamH I and Not I) were purchased from NEB(USA).Lipofectamine2000 and pcDNA3.1/Neo(+) were purchased from Invitrogen .2. To ensure most of the cells were able to grow adhering to the wall at 72hpi, a multiplicity of infection (MOI) of 0.001 was used , which is essential for EIF5A function . The proPlatelet-activating factor acetylhydrolaze (PAFAH) can inactivate the potent pro-inflammatory phospholipid platelet-activating factor (PAF) by removing the acetyl group at its sn-2 position. This family of enzymes is comprised of one secreted (plasma) and at least four intracellular isozymes  ,46. IsofOur study showed platelet-activating factor acetylhydrolaseⅠb subunit beta (PAFAH1B2)to be significantly down-regulated at 72h in H7N9-infected A549 cells. The level of PAF would be up-regulated, which was related to the later clinical symptoms of patients infected with H7N9 virus, such as pneumonia, ARDS, encephalopathy and so on. Moreover, the increase of PAF induces large amounts of platelets to aggregation which can produce a wide range of micro thrombus in the host. It maybe the reason that the patients infected with H7N9 virus exhibit thrombocytopenia and coagulopathy ,9,11.We utilized proteomic approaches to identify differential expressions of cellular proteins in A549 cells infected with H7N9 and H1N1pdm09 influenza virus at 24, 48, 72hpi. It suggests that these differentially expressed proteins are involved in the pathogenic mechanisms of H7N9 infection at the cellular level. A study by Simon et al  infected


We determine the crystal structures of six NAMPT mutants in the apo form and in complex with various inhibitors and use cellular, biochemical and structural data to elucidate two resistance mechanisms. One is the surprising finding of allosteric modulation by mutation of residue Ser165, resulting in unwinding of an α-helix that binds the NAMPT substrate 5-phosphoribosyl-1-pyrophosphate (PRPP). The other mechanism is orthosteric blocking of inhibitor binding by mutations of Gly217. Furthermore, by evaluating a panel of diverse small molecule inhibitors, we unravel inhibitor structure activity relationships on the mutant enzymes. These results provide valuable insights into the design of next generation NAMPT inhibitors that offer improved therapeutic potential by evading certain mechanisms of resistance.Inhibiting NAD biosynthesis by blocking the function of nicotinamide phosphoribosyl transferase (NAMPT) is an attractive therapeutic strategy for targeting tumor metabolism. However, the development of drug resistance commonly limits the efficacy of cancer therapeutics. This study identifies mutations in NAMPT that confer resistance to a novel NAMPT inhibitor, GNE-618, in cell culture and  There is considerable interest in using inhibitors of nicotinamide phosphoribosyl transferase  for cancer therapy Crystal structures of NAMPT in numerous ligand-bound forms have been reported NAMPT mutations that confer resistance to GMX1778, APO866 or TP201565, a structural analog of GMX1778 have been mapped to G217R, H191R, D93del, and Q388R 50 of the corresponding NAPRT1 deficient parental cell lines  or the NAPRT1 proficient NCI-H520 cell line. Short tandem repeat (STR) profiling of the resistant cell lines matched the parental cell lines (data not shown), indicating that they were derived from that cell line.GNE-618 is a novel small molecule that potently inhibits NAMPT activity and exhibits efficacy in xenograft models of cancer NAPRT1 deficient cell lines did not re-express NAPRT1 even when grown in the presence of 10 uM NA, suggesting that this mechanism of resistance is not common in cultured cells. We observe an increase in NAMPT levels in some cell lines, most notably in the RD cell line, consistent with reports that higher levels of NAMPT correlate with decreased sensitivity to NAMPT inhibitors NAMPT from resistant cell lines identified G217R and D93 deletion as well as four novel mutations, G217A, G217V, S165F, and S165Y  for 7 continuous days (QD) by oral administration resulted in 54% tumor growth inhibition . The dosE. coli expression constructs, purified the mutant proteins and evaluated response to GNE-618, APO866 and GMX1778. The H191R and all G217 mutant NAMPT proteins exhibited at least 100- fold increases in GNE-618 IC50 compared to wild-type. The effects on GMX1778 and APO866 were more varied, with G217R and H191R exhibiting the largest shifts and G217V and G217A showing more modest shifts in GMX1778 and APO866 IC50 values  adduct of the inhibitors, because it has been shown that the cellular potency of NAMPT inhibitors is dependent on the formation of a pRib adduct and the resulting inhibitory activity of the compound adduct in silico model predicted that H191R would protrude its side chain into the tunnel and sterically block inhibitors like APO866 from binding We next investigated the structural consequences of mutations at H191 and G217. As depicted in 9-11  or Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and stored in a central cell bank. Lines were authenticated by short tandem repeat (STR) and genotyped upon re-expansions and passaged no more than 20 times after thawing. ICin vivo efficacy studies were approved by Genentech's Institutional Animal Care and Use Committee (IACUC) and adhered to the ILAR Guide for the Care and Use of Laboratory Animals. Nine to fourteen week-old female C.B-17 SCID beige mice (Charles River Laboratories) were inoculated subcutaneously with either 5 million NCI-H460 or 5 million NCI-H460 S156Y cells suspended in a 1∶1 ratio of HBSS and phenol red-free matrigel (BD Biosciences). Mice with similarly sized tumors (mean volume 200–300 mm3) were randomized into treatment cohorts. Mice were dosed for daily (QD) 7 days, orally, with vehicle [PEG400/H2O/EtOH ] or GNE-618. Tumor sizes and body weights were recorded twice weekly. Tumors were measured using digital calipers and volumes were calculated using the formula lw2×0.5 and reported as mean tumor volume ± SEM. Mice with tumor volumes ≥2000 mm3 or with losses in body weight ≥20% from their weight at the start of treatment were euthanized per IACUC guidelines. Percent tumor growth inhibition (TGI) was calculated using the formula [(mean tumor volumevehicle − mean tumor volumetreatment) ÷ mean tumor volumevehicle] ×100. Student's t-test was used to calculate significance in anti-tumor response between drug and vehicle treated mice.All E. coli. Expression was induced in Rosetta (DE3) cells at 260 rpm and 22°C overnight using 1.0 mM IPTG. Cells were harvested by centrifugation at 4000g for 15 min. at 4°C.An expression construct for full length human (XP_540386) NAMPT was constructed using pET21B, a gift from professor Yigong Shi's group . Mutations at positions S165F, S165Y, H191R, G217A, G217R, and G217V were introduced by site-directed mutagenesis. Wild-type and mutant proteins were expressed as C-terminal His6 fusion proteins in Cell pellets were suspended in Lysis buffer A phosphine hydrochloride) and 1X protease inhibitor cocktail Complete (Roche). The Cell suspension was disrupted by passing through a microfluidizer (Model 110F Microfuidizer Cooperator) three times. The whole cell lysate was centrifuged at 40,000 rpm (Beckman Ti45 rotor) for 1 hour at 4°C. The supernatant was applied to a Ni-NTA SuperFlow column (Qiagen), washed extensively with wash buffer B . NAMPT was eluted with elution buffer C . Fractions containing NAMPT were further purified with Superdex 200 column (Amersham) pre-equilibrated with buffer D . The purified NAMPT was greater than 95% pure assessed by SDS-PAGE. The concentration of the purified protein was determined with Bradford protein assay.www.graphpad.com). A mass spectrometry-based assay was used to measure the NAMPT reaction product (NMN) and the internal control (L-Cystathionine) using an Agilent RapidFire/MS system. Analytes were bound to a graphitic carbon cartridge in 0.1% formic acid, eluted in 30% acetonitrile buffer, and injected into a Sciex 4000 mass spectrometer. The analytes in the sample were ionized with electrospray ionization and the positive ions were detected. The Q1 (parent ion) and Q3 (fragment ion) masses of NMN were 334.2 and 123.2, respectively. The Q1 and Q3 for L-Cystathionine were 223.1 and 134.1, respectively. Phospho-Ribose adduct formation assays and NAMPT enzymatic reactions were performed as previously describedNAMPT enzymatic reactions were carried out in Buffer A phosphine (THP)). For compound inhibition studies the final concentration of substrates was 1 µM NAM, 100 µM PRPP, and 2.5 mM ATP. To obtain sufficient NMN levels for detection, we used WT, G217V, G217R, G217A, S165F, and H191R protein at a final concentration of 30 nM, and S165Y protein at 50 nM. 89 µL of enzyme was added to 1 µL 100X compound in DMSO, incubated for 15 minutes at room temperature, the reaction was started with the addition of 10 µL10x substrate solution. After 40 minutes, the reaction was quenched with 10 µL of 10% formic acid and 100 µM L-Cystathionine. Normalized data were analyzed using GraphPad Prism  in LB media containing 50 µg/mL of carbenicillin and then transferred to 22°C prior to induction with 1mM IPTG at an absorbance of 0.8 (OD600). Cells were harvested 16 hours post induction and the pellet was lysed by passing through a microfluidizer in a buffer containing 20 mM TrisCl, pH 8.0, 200 mM NaCl, 5 mM Imidazole, 1 mM TCEP and EDTA-free protease inhibitor tablets (Roche). Cell lysates were loaded onto a HisTrap HP column  in a buffer containing 20 mM TrisCl, pH 8.0, 500 mM NaCl, 5 mM Imidazole and 1 mM TCEP and the bound NAMPT was eluted with 250 mM Imidazole in the same buffer. The protein was then loaded onto a 5 mL Q HP column  in a buffer containing 25 mM TrisCl, pH 8.0 and 1 mM TCEP and eluted at 300 mM NaCl with a linear gradient of the same buffer and 1.0 M NaCl. The protein was further purified on a size-exclusion Superdex 75 column  in a buffer containing 25 mM TrisCl, pH 8.0, 150 mM NaCl and 5 mM DTT. Fractions containing NAMPT proteins were pooled and concentrated to 6 mg/mL and confirmed by SDS-PAGE showing that the purity was greater than 95%.C-terminal His-tagged NAMPT M1-H149 wild-type and mutant proteins with various single mutations were purified and crystallized by similar methods. M1-H491 constructs were transformed into BL21 codon optimized cells. Cells were grown at 37°C to an absorbance of 0.5  and soaked over night at 19°C. The soaked crystals were then harvested with 15% ethylene glycol in the soaking solution without the PRPP and MgCl.Diffraction quality crystals for all the proteins were grown at 19°C from 0.2 uL+0.2 uL vapor diffusion drops containing 6mg/mL NAMPT, 0.1 M Sodium phosphate, pH 8.6–9.0, 25–29% polyethylene glycol 3350 and 0.2 M NaCl, which were then streak-seeded using Hampton's seeding tool. Plate crystals appeared overnight and harvested for data collection with 15% ethylene glycol in the crystallization solution. For the co-structure of NAMPT with phosphoribosyl adduct of GNE-617, apo crystals were transferred into a buffer containing 0.1 M TrisHCl, 25% polyethylene glycol 3350, 0.2 M NaCl, 5 mM MgClThe diffraction data sets were collected at various beam lines and processed with the HKL2000 package Figure S1Resistant cell lines do not re-express NAPRT1 and exhibit smaller decreases in NAD in response to GNE-618. a) Western blots from whole cell lysates of cell lines selected to grow at the indicated doses of GNE-618 represented as fold over IC50 values shown in 50 of the parental cell line, error bars represent the standard deviation of three replicates, c) percent decrease in cellular NAD, the dashed line marks 90% decrease.(TIF)Click here for additional data file.Figure S2GNE-617 in complex with wild-type and S165F NAMPT. NAMPT protein is depicted in ribbons diagram, with wild-type in brown and S165F in cyan. The ligands, GNE-617, are shown in sticks, with orange from the wild-type structure and blue from the S165F mutant structure.(TIF)Click here for additional data file.


Pichia and an in vitro GALT assay. Product characterization showed GALT5 specifically adds galactose to hydroxyproline in AGP protein backbones. Functions of GALT2 and GALT5 were elucidated by phenotypic analysis of single and double mutant plants. Allelic galt5 and galt2 mutants, and particularly galt2 galt5 double mutants, demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared to wild type. Mutant plants showed pleiotropic growth and development phenotypes , which were most severe in the double mutants. Conditional mutant phenotypes were also observed, including salt-hypersensitive root growth and root tip swelling as well as reduced inhibition of pollen tube growth and root growth in response to β-Yariv reagent. These mutants also phenocopy mutants for an AGP, SOS5, and two cell wall receptor-like kinases, FEI1 and FEI2, which exist in a genetic signaling pathway. In summary, GALT5 and GALT2 function as redundant GALTs that control AGP O-glycosylation, which is essential for normal growth and development.Hydroxyproline-O-galactosyltransferase (GALT) initiates O-glycosylation of arabinogalactan-proteins (AGPs). We previously characterized GALT2 (At4g21060), and now report on functional characterization of GALT5 (At1g74800). GALT5 was identified using heterologous expression in  The fundamental processes that underpin plant growth and development depend crucially on the action and assembly of gene products designed to form the cell wall . Cell waAGPs are ubiquitous in the plant kingdom and are expressed in virtually all cells, either at the cell surface as part of the plasma membrane, the cell wall, or as extracellular secretions –9. AGPs O-galactosyltransferase (GALT2), one β-1,3-galactosyltransferase (At1g77810), one β-1,6-galactosyltransferase with elongation activity (GALT31A), one β-1,6-galactosyltransferase with branch initiation and branch elongating activities (GALT29A), and three β-1,6-gluronosyltransferases  . I. I15]. IlcAT14C) –22. FUT4lcAT14C)  identifiArabibopsis) , , –26. Mostibopsis) , 27]. G. GArabibO-galactosyltransferase (Hyp-GALT) that adds the first galactose onto the peptidyl Hyp residues in the AGP core protein is the first committed step in AG polysaccharide addition and represents an ideal control point to investigate the contribution of AG polysaccharides to AGP function. Previously, GALT2 (At4g21060) was demonstrated to encode a Hyp-GALT Gal. The 14C-radiolabel incorporation was estimated by resuspending the pellets in 300 μl of water before counting in a liquid scintillation counter.A standard GALT assay was performed using 20 μg of various peptide substrate acceptors, (AO)g et al. . Rhamnog7 peptide as the acceptor substrate. Assay products from each reaction were fractionated by RP-HPLC to measure incorporated 14C-radiolabel into acceptor substrates. The optimum pH for GALT5 activity was determined using permeabilized microsomal membranes (250 μg) from the C5 Pichia line expressing His6-GALT5 dissolved in test buffers at a final concentration of 100 mM. Test buffers included MES-KOH buffer at pH 4, 5, 6, and 7; HEPES-KOH buffer at pH 6, 6.5, 7, 7.5, and 8; Tris-HCl buffer at pH 8, 9, and 10; and CAPS-KOH buffer at pH 9 and 10.The standard GALT assay was modified for GALT5 characterization using (AO)2, MgCl2, CaCl2, CuCl2, NiCl2, or ZnSO4 was added to the GALT assay  when tested. Two controls were added, one with no ions in the buffer used for resuspending the detergent permealized membrane fraction and the other with EDTA (5 mM) to chelate any residual divalent cations trapped in the membranes. An equal volume of deionized distilled water was added instead of divalent ions in the control reaction.To examine the effect of divalent cations on GALT5 activity, microsomal membranes were extracted with homogenizing buffer lacking divalent ions. MnCl7 as the acceptor substrate and various 14C-radiolabeled nucleotide sugar donors . The nucleotide sugars tested included UDP-[14C]Glc , UDP-[14C]Xyl (PerkinElmer Life Sciences), and GDP-[14C]Fuc (PerkinElmer Life Sciences). Four separate GALT reactions with no substrate acceptors were performed as controls.To analyze the enzyme specificity for nucleotide sugar donors, the standard activity assay was performed with (AO)GALT5 coding region was subcloned into pEarleyGate 101 plasmid to generate the GALT5:YFP construct by a gateway cloning strategy. The primers used in cloning are listed in Agrobacterium-mediated transient expression was performed in the leaves of three to four week-old tobacco plants (Nicotiana tabacum cv. Petit Havana) grown at 22–24°C using a bacterial optical density (OD 600) of 0.05 for single infiltrations and 0.025 each for co-infiltrations 7 as the peptide substrate acceptor and UDP-[14C]Gal as the sugar donor.Plant microsomal membranes were extracted according to Liang et al.  with mingalt2-1, galt2-2, galt5-1, galt5-2, and galt2 galt5 mutant plants as described in Schultz et al. Gal as the sugar donor and one of two AGP peptide analogs (d[AO]51 and [AO]7) as the substrate acceptor. The amount of GALT activity varied in the five recombinant cloned cell lines (C1 to C5) of Pichia based on the rate of [14C]Gal incorporation using the [AO]7 substrate acceptor, but all were significantly higher than Pichia cells transformed with the vector alone, which served as a negative control (NC)  .Pichia transformants expressing GALT5 were further analyzed for Hyp-GALT activity using two substrate acceptors: [AO]7, a synthetic AGP peptide and d[AO]51, a transgenically expressed and chemically deglycosylated AGP analog. Incorporation of [14C]Gal from UDP-[14C]Gal onto the two substrate acceptors was observed by HPLC fractionation    Fig  and 1F. tosidase  or be coeviously . Peak I eviously . Microso(NC) Fig  and 1E. 14C]radiolabel remained associated with Gal, RP-HPLC fractions containing the [14C]radiolabeled [AO]7:GALT5 reaction products were pooled and subjected to total acid hydrolysis. The resulting acid hydrolyzed [14C]radiolabeled monosaccharide was fractionated by HPAEC and showed that the [14C]label co-eluted with Gal, thereby confirming incorporation of [14C]Gal onto the [AO]7 peptide  on the P2 column, whereas the base hydrolysate of this product eluted at DP4  and Golgi Predictor http://ccb.imb.uq.edu.au/golgi/) and the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/) ,  a  a 60] a galt2 or galt5 mutants are largely indistinguishable from wild type with respect to their non-biochemical phenotypes, galt2 galt5 double mutants are clearly compromised with respect to normal growth and development  lead to impaired pollen tube elongation . Si. Sigalt t growth , 71]. I. Igalt msos5 mutants are known to be salt hypersensitive and exhibit root tip swelling 7-dependent GALT activity tests of the five transgenic Pichia cell lines using Triton X-100 permeablized microsomal membranes. For each line, 250 μg of total microsomal membrane protein was used for the assay. [14C]Gal radiolabel incorporation is expressed as pmol/h/mg protein and reflects the difference between total incorporation obtained in reaction products in the presence versus absence of [AO]7 acceptor substrate. Reactions were done in triplicate and mean values ±SE are presented. All cell lines tested had GALT5 activity but varied in the rate of incorporation. Student’s t-test was performed using Graphpad Quickcalc  and significant differences in GALT activity were detected with respect to NC .(A) Western blot analysis of the 6x His-tagged GALT5 protein in (EPS)Click here for additional data file.S3 FigPichia C5 line expressing 6x His-GALT5 served as the enzyme source in the [AO]7:GALT reaction. The [14C]-labeled monosaccharides were analyzed by High-Performance Anion-Exchange Chromatography (HPAEC) on a CarboPac PA-20 column. Elution times of monosaccharide standards are as indicated with arrows at the top.Permeablized microsomal membranes from the transgenic (EPS)Click here for additional data file.S4 Fig14C]radiolabeled galactose onto [AO]7 substrate acceptor in presence of UDP-[14C]Glc, UDP-[14C]Gal, UDP-[14C]Xyl, and GDP-[14C]Fuc. (B) Effect of pH on enzyme activity. (C) Effect of different divalent ions (5 mM) on enzyme activity.Data presented are an average of duplicate assays. A. Specificity of the GALT5 enzyme for nucleotide sugar donors was analyzed by monitoring incorporation of (EPS)Click here for additional data file.S7 Figgalt2galt5 root hairs (B) pollen tubes and (C) seeds. Significant reduction in signal intensity was observed in the galt2galt5 samples compared to the WT. All immune-histochemical experiments were repeated twice with ten seedlings for root hairs, ten flowers with five pollen tubes and 50 seeds for each genotype respectively.(A) Confocal microscopy images of WT and (EPS)Click here for additional data file.S8 Figin vitro on MS media (B) and adult plants grown in soil during long days. Key developmental stages were monitored according to Boyes et al. [(C) Plants at 28 d after germination.(A) Phenotypes of the indicated seedlings grown s et al. . Data pr(EPS)Click here for additional data file.S9 Figin vitro germination of pollen in the germination media. WT pollen tubes did not display tube disruption and were used as a control. Bar = 50 μm.Disruption of tip growth in pollen tubes as observed after 8 h of (EPS)Click here for additional data file.S10 Figgalt2, galt5 and galt2 galt5 seeds on various concentrations of NaCl. Seeds were stratified at 4°C in the dark for 48 h and then transferred at 22°C under continuous white light and were quantified after 5 d. Data are the mean ± SE of three biological replicates according to a Student's t test .Germination frequencies of WT, (EPS)Click here for additional data file.S11 Figgalt2, galt5 and galt2 galt5 seeds were germinated on MS medium supplemented with either 100 mM NaCl (A) or 150 mM NaCl (B). Radicle lengths were measured over time and standard errors were calculated from at least three independent experiments, each of which contained 50 seeds of WT and 50 seeds of each mutant. Emergence of the radicle was considered the indication of germination. Radicle length was measured by Motic Image version 3.2.WT, (EPS)Click here for additional data file.S12 Figgalt2-1, galt2-2, galt5-1, galt5-2 and galt2 galt5 were grown on MS medium with 1% sucrose for 3 d. Seedlings were then transferred to MS plates supplemented with 100 mM NaCl, and photographed after 7 d (A), 14 d (B) and 21 d (C). Bar = 1cm.Five-day-old seedlings of WT, (EPS)Click here for additional data file.S13 Figgalt2-1, galt2-2, galt5-1, and galt5-2 seeds were grown on MS medium with 1% sucrose for 5 d. Root growth was measured 7 d after transferring the seedlings to MS plates supplemented with various concentrations of (A) NaCl, (B) KCl, (C) LiCl, (D) CsCl, and (E) mannitol. Error bars indicate SEs (n = 25). The experiments were repeated at least three times with similar results.WT, (EPS)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Anopheles funestus is rapidly expanding across Southern Africa. It remains unknown whether this resistance has a unique origin with the same molecular basis or is multifactorial. Knowledge of the origin, mechanisms and evolution of resistance are crucial to designing successful resistance management strategies.Pyrethroid resistance in the major malaria vector An. funestus population at the northern range of the resistance front. Similar to other Southern African populations, Zambian An. funestus mosquitoes are resistant to pyrethroids and carbamate, but in contrast to populations in Mozambique and Malawi, these insects are also DDT resistant. Genome-wide microarray-based transcriptional profiling and qRT-PCR revealed that the cytochrome P450 gene CYP6M7 is responsible for extending pyrethroid resistance northwards. Indeed, CYP6M7 is more over-expressed in Zambia  than CYP6P9a  and CYP6P9b , whereas CYP6P9a and CYP6P9b are more highly over-expressed in Malawi and Mozambique. Transgenic expression of CYP6M7 in Drosophila melanogaster coupled with in vitro assays using recombinant enzymes and assessments of kinetic properties demonstrated that CYP6M7 is as efficient as CYP6P9a and CYP6P9b in conferring pyrethroid resistance. Polymorphism patterns demonstrate that these genes are under contrasting selection forces: the exceptionally diverse CYP6M7 likely evolves neutrally, whereas CYP6P9a and CYP6P9b are directionally selected. The higher variability of CYP6P9a and CYP6P9b observed in Zambia supports their lesser role in resistance in this country.Here, we established the resistance profile of a Zambian Pyrethroid resistance in Southern Africa probably has multiple origins under different evolutionary forces, which may necessitate the design of different resistance management strategies.The online version of this article (doi:10.1186/1471-2164-15-817) contains supplementary material, which is available to authorized users. Anopheles funestus, is threatening the effectiveness of these control tools across Africa  by Genetic Services  to generate the transgenic line UAS-CYP6M7. Ubiquitous expression of the transgene CYP6M7 in adult F1 progeny  was obtained after crossing virgin females from the driver strain Act5C-GAL4 .The higher genetic diversity of s Africa . Very los Africa . A similAn. funestus. Indeed, this study has revealed that beside the previously known resistance genes CYP6P9a and CYP6P9b, the CYP6M7 P450 gene is also playing a key role in the pyrethroid resistance observed in the region and that its role may even become more important in the northern range of the resistance front such as in Zambia. Therefore, despite the similarities observed, the underlying molecular basis of pyrethroid resistance is probably not uniform across its distribution range in Southern Africa. The northern range in Zambia exhibits significant differences from Malawi and Mozambique in term of the profile and molecular basis of resistance. It is therefore likely that beyond Mozambique and Malawi, pyrethroid resistance in southern Africa has multiple origins under different evolutionary forces, which may necessitate the design of different resistance management strategies to mitigate the impact of this resistance.Taken together, the results from this study have helped to shed more light into the molecular complexities of the resistance mechanisms underlying pyrethroid resistance in southern African populations of Table S2. Top 50 probes the most commonly up-regulated in R-S in Mozambique, Malawi and Zambia. Table S3. Top 20 probes the most commonly down-regulated in R-S in Mozambique, Malawi and Zambia. Table S4. Summary statistics for polymorphism of CYP6M7 between susceptible and resistant mosquitoes in Zambia, Malawi and Mozambique. Table S5. Codon-based Test of Selection for CYP6M7 for analysis averaging over all sequence pairs within each group. Table S6. Summary statistics for polymorphism of CYP6P9a between susceptible and resistant mosquitoes in Zambia, Malawi and Mozambique. Table S7. Summary statistics for polymorphism of CYP6P9b between susceptible and resistant mosquitoes in Zambia, Malawi and Mozambique. Table S8. Genetic differentiation using KST for CYP6P9a, CYP6P9b and CYP6M7. (PDF 315 KB)Additional file 1: Table S1: List of primers used in this study. Figure S2. Maximum likelihood (ML) tree of full-length CYP6M7 cDNA haplotypes from various regions of Africa. Figure S3. Analysis of polymorphisms of CYP6M7, CYP6P9a and CYP6P9b. Figure S4. Protein variants among resistant and susceptible mosquitoes from all three countries. Figure S5. Phylogenetic analysis of CYP6M7. Figure S6. Impact of permethrin resistance on genetic diversity. Figure S7. Comparative analysis of the haplotypes of the three genes based only on coding regions among susceptible and resistant mosquitoes from the three countries using a 95% parsimony network. (PDF 638 KB)Additional file 2: Figure S1: Gene expression analysis.


At the heart of all contemporary biochemical systems reside reactive phosphorus (P) molecules  as energy currency molecules to drive endergonic metabolic processes and it has been proposed that a predecessor of such molecules could have been pyrophosphate . Arguably the most geologically plausible route to PPi(V) is dehydration of orthophosphate, Pi(V), normally a highly endergonic process in the absence of mechanisms for activating Pi(V). One possible solution to this problem recognizes the presence of reactive-P containing mineral phases, such as schreibersite [3P] within meteorites whose abundance on the early Earth would likely have been significant during a putative Hadean-Archean heavy bombardment. Here, we propose that the reduced oxidation state P-oxyacid, H-phosphite  could have activated Pi(V) towards condensation via the intermediacy of the condensed oxyacid pyrophosphite . We provide geologically plausible provenance for PPi(III) along with evidence of its ability to activate Pi(V) towards PPi(V) formation under mild conditions (80 °C) in water.The emergence of mechanisms for phosphorylating organic and inorganic molecules is a key step  In this contribution we provide geologically plausible provenance for both Pi(III) and PPi(III) along with evidence of the ability of the latter to provide a low energy route to PPi(V) via activation of Pi(V) towards condensation in the presence of divalent metals.Amongst the most important and ubiquitous energy-currency molecules of contemporary biochemistry are activated phosphorus (P) species such as phosphocreatine, phosphoenol pyruvate, and adenosine triphosphate (ATP) . These meactions  and subseactions . A signir of ATP ,6, not ictions 2O4− has ber of ATP . Not onlr of ATP . We presr of ATP , would hr of ATP , energy r of ATP  to use tr of ATP  and a rehemistry  and lowehemistry  and it hhemistry , althoughemistry . Here, w2O for NMR analyses was used as received from Sigma-Aldrich. Solutions of aqueous HCl, NaOH and Na2S were prepared by dilution of commercial samples in PSA deionized water. Solution pH measurements were made on a Schochem pH meter buffered to pH 4 and 7 with commercial  standards. 31P-NMR analyses were performed on a Bruker Avance 500 MHz instrument operating at 202.634 MHz for 31P internally referenced to 85% H3PO4. Iron, principally in the form of ferrous (Fe2+) for our Icelandic field samples, was removed from all samples prior to NMR analysis to alleviate the problems associated with paramagnetic broadening. This was done by pH adjustment, first to ca. 12 by addition of NaOH (aq) (1 M) which leads to precipitation of oxides of iron, followed by addition of aqueous Na2S solution (1 M), centrifugation, filtration and re-adjustment back to pH ca. 4 with HCl (1 M). For each sample, 10 mL of fluid were reduced to dryness and the residue redissolved in 0.5 mL deionized water or D2O, filtered using 0.45 μm syringe filters and analyzed. For those samples run in H2O solvent, D2O inserts were used to provide a deuterium lock. Samples within which pyrophosphite, PPi(III), was expected to be present and analyzed were pH adjusted to between 7–8 by addition of NaOHaq (1 M).Water was purified by ion exchange on a Purite Select Analyst (PSA) reverse osmosis-deionization system . D3PO3 (50 mM) in deionized water were pH adjusted to 4.5, 5.0, 5.5, and 5.8 with aqueous NaOH (50 mM) solution before being evaporated to dryness, dried for several h in a Belling open oven at 50 °C, ground to a fine powder and then placed in a vacuum oven set to 85 °C, 1–10 mbar pressure for 4, 5, and 6 days. After this time, an aliquot (ca. 50 mg) was removed, dissolved in distilled water, pH adjusted by addition of NaOH (1 M) solution to between 7–8 to mitigate against acid-catalyzed hydrolysis of PPi(III) and analyzed, in triplicate, by 31P-NMR spectroscopy (data are collected in 3PO3 (0.1 M) in standard mean ocean water  were pH adjusted to 3.0 and 4.0 with aqueous NaOH (0.1 M) solution before being evaporated to dryness, dried for several h in a Belling open oven at 50 °C, and then placed in a vacuum oven set to 85 °C, 1–10 mbar pressure for several days (up to 2 weeks). After this time, an aliquot (ca. 50 mg) was removed, dissolved in distilled water, pH adjusted by addition of NaOH (1 M) solution to between 7–8 to mitigate against acid-catalyzed hydrolysis of PPi(III) and analyzed by 31P-NMR spectroscopy in triplicate  for various time periods under different pH and temperature conditions. Samples A1, 3, 5, 6, 7, 8 were incubated with initially hot fluids for four days but were allowed to cool to ambient temperature and left for between 82–86 days prior to analysis . Samples A2 and 4 were incubated in the fluids noted at the natural temperatures for 4 days (A2) and 2 days (A4) respectively prior to work up for analysis via ICP-AES or ICP-MS methods through a sequence of steps as outlined below:(i)Samples were pre-filtered using Whatman Number 1 filter papers.(ii)Samples were then diluted to 70 mL and re-filtered using Pall Corporation Acrodisc 32 mm 0.45 µm syringe filters to remove un-dissolved particulates.(iii)Nalgene bottles (30 mL capacity) were acid washed by filling with 50% HCl and leaving for 24 h. They were rinsed 3 times with tap water and 3 times with deionized water then left to air dry.(iv)Two portions of 35 mL of sample were placed in an acid-washed 30 mL Nalgene bottle.(v)ca. 2 drops) was added to one of the portions and the lids screwed on tightly and the bottles labeled.Concentrated nitric acid ((vi)Due to the concentration of sulphur, the acid treated samples were further diluted to allow accurate measurement of the sulphur content of each sample.(vii)Two 1.5 mL aliquots were taken from each of the acid treated samples. Two drops of toluene was added as an antibacterial agent. The sub-samples were subsequently submitted for ICP analysis.Eight samples of Fe−1. Three standards were run for P analysis and sample standard deviations included accordingly. Data are collected in ® AS17C chromatographic column preceeded by an AG17 Guard Column. This method represents a new application for ICP-MS and will be discussed in detail in a forthcoming paper. General methods were modified from those used for P speciation on IC-Electro spray MS and optimized for HPLC-ICP-MS. Sample aliquots (50 μL) were eluted with KOH using a linear concentration gradient at 1.5 mL/min flow rate. P was detected on the ICP-MS as mass 31 and the ICP-MS was optimized as before. Calibration standards were made from reagent grade or better synthetic H-phosphorous [identified as P(III)] and orthophosphoric [identified as P(V)] acids and were mixed just prior to analysis. Calibrations were performed on orthophosphate, H-phosphite and hypophosphite at 0.3, 3.0 and 30 μM and calibration curves yielded r2 better than 0.995.Fluids from a variety of sampling sites inthe Hveradalur geothermal area were pre-filtered to remove suspended particulate matter followed by fine-filtration using a Millipore 0.45 μm filter (post-collection in the UK). Duplicate 30 mL water samples were taken, one of which was acidified with nitric acid, and these were analyzed with a Dionex Ion Chromatograph and Horoba JY Ultima 2C ICP-AES for dissolved anion and cations respectively, at the Wolfson Geochemistry Laboratory at Birkbeck College—UCL. Cation results were taken as the mean of three repeat measurements, with standard deviations typically between 0.01–0.1 mg L31P-NMR analysis were prepared as follows. Pre-heated samples were analyzed after steps (i)–(iv) and post-heated subsequently after steps (vi)–(ix).(i)A 10 mL aliquot of the acidified sample (prepared as above) was taken in a 15 mL Falcon tube.(ii)Sample was treated with NaOH (1 M) to pH 12 and left for 1–2 min.(iii)Sample was gravity filtered to remove precipitate (hydrated ferrous oxides and hydroxides). These oxides were subsequently collected and shown to contain negligible amounts of phosphorus via EDX measurements.(iv)Sample was treated with HCl (1 M) to pH 4.(v)31P-NMR spectroscopy  using capillary D2O inserts.A 0.5 mL aliquot was taken and analyzed by (vi)Sample was reduced to dryness and residues dried overnight in 50 °C oven.(vii)Residues were ground to fine powder in mortar and pestle.(viii)ca. 90 °C for 72 h on a sand bath under flowing N2 (ca. 1 bubble per second).Residues were dry heated to (ix)ca. 0.5 mL) and adjusted to pH 7.2 using aqueous Na2CO3 solution (1 M).Residues were dissolved in deionized water ((x)31P-NMR spectroscopy  using capillary D2O inserts.Sample was analyzed by All Iceland samples for 31P-NMR responses, we presume due to the presence of trace amounts of iron in the system. Nevertheless, heated samples A1 and A5 show clear PPi(III) present, confirmed by addition of authentic samples and there is evidence for PPi(III) presence in the remaining three A samples 2, 3, and 4. Samples A6–8 did not show any PPi(III) after the above heating protocol but we do have evidence that there are several factors that affect PPi(III) production in the solid phase such as degree of dryness and particle size of the heated material. These and other variables in the solid phase dehydration of Pi(III) to PPi(III) are currently under investigation.All of the pre-heated samples,  reveal t2-PPi(III), reacts readily with aqueous solutions of sodium phosphate to give sodium isohypophosphate, Na2-PPi(III–V) (2-Pi(V)  is dissolved with gentle warming in 10 mL deionized water and allowed to cool to room temperature. To this is added Na2-PPi(III)  and the flask stoppered and allowed to stand in a cool place at temperatures in the range 5–15 °C for 48 h. The PPi(III) does not immediately dissolve. After this time the phosphorus speciation is measured by 31P-NMR spectroscopy. Normally, a few percent PPi(III) remain which can be allowed to hydrolyze by raising the pH to 8 leaving a solution of approximately 46% of total phosphorus being present as PPi(III–V) with the remainder as Pi(III) and Pi(V). In order to reduce the competitive hydrolysis pathway of PPi(III) still further and raise the yield of PPi(III–V), a large excess of Pi(V) can be used as described by Blaser and Worms :Mg of ca. 6:1. If more magnesium is added, then PPi(III–V) formation is accelerated (vide infra).Sodium pyrophosphite, Nai(III–V) . The reand Worms . In a sivide infra) and in lower overall yields. In the absence of magnesium no reaction appears to take place in solution. A solution containing PPi(III–V) 43.5%, Pi(V) 6.9% and Pi(III) 49.5%  was diluted 6-fold to give a stock solution approximately 0.1 M in PPi(III–V). To this was added disodium hydrogen phosphate  and MgCl2·6H2O  thereby making a solution with two equivalents of Pi(V) and three equivalents of Mg2+  with stirring. The precipitates slowly dissolved and the phosphorus speciation was determined using 31P-NMR spectroscopy. The solution contained PPi(V) as 1.7% total phosphorus irrespective of the starting pH being 6 or 7 and all PPi(III–V) had been consumed. Approximately 7% of the PPi(III–V) has been phosphorylated to PPi(V) (PPi(III–V) reacts further with Pi(V) to give PPi(V) but under more forcing conditions ( of Mg2+ a. The soo PPi(V) b. In con2PO4 or CaHPO4; Pi(V)] between 250–600 °C  salts allows dehydration to PPi(III) under far milder conditions. Thus, isothermal thermogravimetric analysis . 31P-NMR analysis of a non-pH adjusted sample of this material confirms PPi(III) formation with a characteristic AA’XX’ spin system  consistently afford PPi(III) in up to 30% after 6 days at 85 °C in the presence of a gas flow (either air or dinitrogen) or within a vacuum oven whereas starting from solutions at pH’s 6 and above show zero PPi(III) formation under the same conditions (upper). Furthermore, dry heating Pi(III) solids from evaporated, simulated ocean water solutions  to 85 °C affords effective conversions to PPi(III) . Moreover, not only is initial solution pH a key factor influencing PPi(III) formation under these conditions, but mechanical action, presumably influencing particle size, and hence water-release, is also important. Thus, grinding dried samples of Pi(III) from distilled water prior to heating at 85 °C under gas flow results in up to 7 times the yield of PPi(III). Under the same conditions, dry heating solids from evaporated aqueous solutions of Pi(V) affords no PPi(V) formation.Pyrophosphate [PPi(V)] can be formed by dry heating orthophosphate salts . F. F2PO4 onditions upper. F3P  after 16 h incubation at pH 7 (2 or CaCl2 (50 mM) greatly enhances PPi(III–V) formation whereupon ca. 80% of total Pi(V) is converted to PPi(III–V) (2+ increase the rate at which PPi(III–V) is formed from PPi(III) and Pi(V), but also appears to accelerate the hydrolysis reaction of the former. This is potentially highly significant since it is points towards both rapid formation of and P-transfer from PPi(III–V), a chemical feature we are currently probing in more detail in this laboratory.Identifying a viable chemical pathway from any primitive energy currency system to one based around PPi(V), can only strengthen the plausibility for the former and several such processes have been proposed ,41,42,43 at pH 7 . Moreovei(III–V) . Intriguactivates the Pi(V) moeity. Thus, to a solution comprising PPi(III–V) 43.5%, Pi(V) 6.9% and Pi(III) 49.5%  was added Na2HPO4 and MgCl2·6H2O to achieve a solution with 2 equivalents of Pi(V) and 3 equivalents of Mg2+ with respect to total P. After heating this mixture to 80 °C in aqueous solution for 42 h, PPi(V) is detected at 1.7% of total P  to PPi(V) under these mild conditions. Such coupling of Pi(V) moieties does not occur under such mild conditions in the absence of PPi(III) or alternative coupling agents , reactions that can be reproduced in both field and laboratory settings. Moreover, we find that PPi(III) is an excellent P-transfer reagent, reacting smoothly with orthophosphate [Pi(V)] in aqueous solution  to afford isohypophosphate, PPi(III–V). In turn, heating solutions of PPi(III–V) with further Pi(V) to 80 °C in aqueous solution, results in exchange of Pi(III) for Pi(V) and formation of pyrophosphate, PPi(V) in overall 7% conversion efficiency from PPi(III–V). This, we contend, provides evidence of the ability of the Pi(V) centre in PPi(III–V) being activated towards substrate-level phosphorylation to afford PPi(V) under relatively mild conditions (80 °C in aqueous solution) given that PPi(V) formation from Pi(V) does not occur under the same conditions in the absence of added PPi(III). Further studies are continuing to probe chemistries of PPi(III) and PPi(III–V) within putative Hadean geological environments.In summary, we have demonstrated here that low pH geothermal fluids, such as those found within sub-glacial volcanic environments, are capable of producing reduced oxidation state P-compounds from schreibersite models (Fe


Stroke is the most serious complication of atrial fibrillation (AF). Of the estimated 600,000 ischemic strokes that occur each year in the United States, 15-20 percent are thought to be secondary to AF. Blood stasis is a key factor responsible for thrombus formation in the left atrium (LA). Studies utilizing transesophageal echocardiography have shown that peak left atrial appendage flow velocity < 0.2 m/s is an independent risk factor for stroke in AF. 4D Flow MRI may improve prediction of stroke risk in AF patients by examining 3D flow patterns across the entire LA volume over multiple phases spanning the cardiac cycle. We hypothesized that LA stasis measured by 4D flow MRI correlates with stroke risk estimated by the CHA2DS2-VASc clinical risk score in patients with AF.We performed 4D flow MRI in 31 patients with documented AF, 21 were in sinus rhythm at the time of imaging (AF-sinus) and 10 were still in AF (AF-afib). Each subject underwent standard cardiac protocols on 1.5T and 3T MR systems  including CINE images to determine cardiac function. In addition, ECG and navigator gated free breathing 4D-Flow MRI was performed for each subject . Data analysis included noise filtering as well as correction for Maxwell terms, eddy currents, and velocity aliasing. LA volume was manually segmented to enable analysis of all LA voxel velocities over multiple phases spanning the cardiac cycle. The top two tertiles of LA velocities were used for analysis, although results were similar if 100% of the velocities were used. LA flow for each patient was quantified by mean LA velocity and the percentage of LA velocities > 0.2 m/s (incidence). CHA2DS2-VASc scores were calculated for each patient .Patient age was 59.7 ± 9.4 in AF-sinus and 68.1 ± 8.6 AF-afib. CHA2DS2-VASc correlated negatively with LA mean velocity  and the American Heart Association (12GRNT12080032 - MM).


Listeria monocytogenes Lm60, a fast cold-adapting serotype 1/2a human isolate, is presented.The complete genome sequence of  Listeria monocytogenes is an important food-borne pathogen associated with serious illness and high mortality in those with weakened immunity  at the Functional Genomics Centre at the University of Zurich. Sequencing resulted in 27, 714 sequence reads (48-fold genome coverage) with an average length of 5,854 kb. The Lm60 genome was assembled de novo using the SMRT Analysis 2.1.1 software and HGAP3 algorithm to a single chromosome of 2,989,591 bp in size with a G+C content of 38%. Gene prediction and annotation was carried out using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline.We present here the complete genome sequence of in silico multilocus sequence type (MLST) analysis was performed using the MLST targets  and Lm60 was assigned to sequence type (ST) 551 (http://www.pasteur.fr/recherche/genopole/PF8/mlst/index.html).The Lm60 genome contains a total of 3,002 genes including 2,904 coding sequences (CDS), 12 pseudogenes, 67 tRNA genes, and 6 16S-5S-23S operons. Using the Phage search tool (PHAST) , the Lm6(ST) 551  (http://L. monocytogenes strains.The availability of the genome sequence from this strain will provide insight into possible genetic mechanisms associated with enhanced cold stress resistance among fast cold-adapting CP009258.The complete Lm60 genome has been deposited in GenBank under the accession no.