In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease.ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein  confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity.  Human ATP-binding cassette (ABC)Considerable efforts have been made to achieve two clinical goals: 1) turn off ABC drug pumps such P-gp (ABCB1), BCRP (ABCG2), and MRP1 (ABCC1) to prevent multidrug resistance; and 2) repair folding mutants of CFTR , BCRP , ABCB4/ABCB11 , ABCA1 (Tangier disease), ABCC2 (Dubin-Johnson syndrome), ABCC6 (pseudoxanthoma elasticum), and ABCC8 (hyperinsulinimic hypoglycemia of infancy) using small molecules called correctors or pharmacological chaperones. Large scale screening efforts have been promising but most compounds identified to date have been poor therapeutic candidates as they only caused partial inhibition of the pump or rescue of misfolded ABC proteins.Studying P-gp interaction with tariquidar is important for understanding how to turn off drug pumps or rescue misfolded mutants. Tariquidar is different from previous inhibitors because it is not transported by P-gp but binds to the protein to activate ATPase activity . Althougm tariquidar   , Caenorh elegans . There iw errors –17. In s docking  and mole docking  have rec docking . For exa  . A problHere, we used cross-linking protection assays and arginine mutagenesis of residues within the 12 TM segments to test for residues close to or within the tariquidar-binding site. The rationale for arginine mutagenesis was that insertion of a bulky charged side chain into a tariquidar-binding site would inhibit tariquidar rescue of processing mutants and tariquidar-stimulated ATPase activity. Our results suggest that tariquidar binds to a site within the drug-binding pocket because 30 arginines introduced into the TM segments disrupted both tariquidar rescue of processing mutants and tariquidar-stimulated ATPase activity. Unlike other drug substrates, tariquidar promoted maturation and stabilized the first transmembrane domain (TMD1). Stabilization of TMD1 may be an important mechanism in rescuing misfolded ABC proteins because a similar mechanism appears to be involved in the rescue of misprocessed CFTR protein by the corrector VX-809 .55-Phe72 (TM1), Ser119-Cys137 (TM2), Lys189-Val206 (TM3), Leu214-Trp232 (TM4), Thr294-Ala311 (TM5), Val331-Ala348 (TM6), Val712-Phe732 (TM7), Phe759-Phe777 (TM8), Lue833-Ser850 (TM9), Leu857-Val874 (TM10), Phe938-Gly955 (TM11), or Val974-Ser992 (TM12). Mutants were constructed to contain an A52 epitope tag at their C-terminal ends for use in whole cell immunoblot assays  containing the A52-epitope or 10-histidine tags  by site-When an arginine mutation was identified that inhibited rescue with tariquidar, it was then introduced into the histidine-tagged wild-type P-gp cDNA. The P-gp cDNA was modified to contain a 10-histidine tag at the COOH-terminal end to facilitate purification of the expressed protein by nickel-chelate chromatography . Truncatm CaCl2 was added to 90 μl of H2O containing 2 μg of DNA followed by addition of 100 μl of BES solution . After 10 min at room temperature, 4 ml of HEK 293 cells  in Dulbecco's modified Eagle's medium (DMEM) with high glucose  bovine calf serum) was added and 1.5 ml of the mixtures were added to duplicate well of 6-well culture plates. After 4 h at 37 °C, the medium was replaced with fresh medium with or without 0.5 μm tariquidar (UHN (Shanghai) Research & Development Co., Toronto, ON). About 16 h later, the cells were harvested, washed with PBS, and cell pellets were suspended in 150 μl of 2× SDS sample buffer  SDS, 4% (v/v) 2-mercaptoethanol containing 25 mm EDTA). Samples were applied to 6.5% SDS-PAGE gels . The gels were electroblotted onto a sheet of nitrocellulose and P-gp proteins were detected using A52 monoclonal antibody, horseradish peroxidase-conjugated anti-mouse secondary antibody, and enhanced chemiluminescence . An equivalent amount of the sample was also loaded onto 10% (v/v) SDS-PAGE gels and subjected to immunoblot analysis with a monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GADPH) . The signals were imaged and relative levels of immature (150 kDa) and mature (170 kDa) P-gp were determined using ChemidocTM XRS+ with Image LabTM software (Bio-Rad).The G251V mutant cDNAs were transiently expressed in HEK 293 cells by a calcium phosphate precipitation approach as described previously . Brieflym dithiothreitol before membrane preparation because the mutant cross-links spontaneously (m copper phenanthroline (oxidant to promote disulfide bond formation) in the presence or absence of 0.25 μm (for TM segment cysteine mutants) or 1 μm tariquidar (for the ICL and NBD cysteine mutants). Mutant T333C/L975C was cross-linked in whole cells at 20 °C with 0.5 mm BMOE  cross-linker  in the absence or presence of 0.25 μm tariquidar  in the absence or presence of 1 μm tariquidar  glycerol, and 4% (w/v) SDS) containing 25 mm EDTA and no reducing agent. The reaction mixtures were then subjected to SDS-PAGE (6.5% (w/v) polyacrylamide gels and 7% gels for mutant A80C/R741C) and immunoblot analysis with a rabbit polyclonal antibody against P-gp. Intramolecular disulfide cross-linking between domains can be detected because the cross-linked product migrates with a slower mobility on SDS-PAGE gels (Cys-less P-gp mutants containing pairs of cross-linkable cysteines in the TM segments (A80C(TM1)/R741C(TM7), I299C(TM5)/F770C(TM8), and T333C(TM6)/L975C(TM12)), intracellular loops (ICLs) (L175C(ICL1)/N820C(ICL3), A259C(ICL2)/W803C(ICL3), and A266C(ICL2)/F1086C(NBD2)), or the NBDs (C431(NBD1)/L1176C(NBD2), L531C(NBD1)/C1074(NBD2), and P517C(NBD1)/I1050C(NBD2)) were transiently expressed in HEK 293 cells at reduced temperature 30 °C) to promote maturation. Cells expressing mutant A80C(TM1)/R741C(TM7) were washed with PBS containing 10 m °C to prm cyclosporine A at 37 °C in the presence of 5 mm sodium butyrate for 24 h and then incubated at 30 °C for 24 h. Sodium butyrate is a histone deacetylase inhibitor that enhances expression of proteins in HEK 293 cells  SDS, 100 mm EDTA, and 10% (v/v) 2-mercaptoethanol). For treatment with endoglycosidase Hf , 0.1 volume of 0.5 m sodium citrate, pH 5.5, was added to a sample of the solubilized cells followed by addition of endoglycosidase H to a final concentration of 20,000 units/ml. For treatment with endoglycosidase F , 0.1 volume of 0.5 m sodium phosphate buffer, pH 7.5, and 10% (v/v) Triton X-100 were added to sample of the solubilized cells followed by addition of PNGase F to a final concentration of 10,000 units/ml. The samples were incubated for 15 min at 37 °C and the reactions stopped by addition of 1 volume of 2× SDS buffer. The samples were run on 10% SDS-PAGE gels and subjected to immunoblot analysis.In experiments involving endoglycosidase digestion, cells were co-transfected with cDNAs of A52-tagged TMD1 and untagged TMD2 and expressed in the presence of 1 μm tariquidar. The membranes were suspended in Tris-buffered saline, pH 7.4 (5 mg/ml of protein). Samples of the membranes were treated for 5 min at 20 °C with various concentrations of TPCK-trypsin . The reactions were stopped by addition of lima bean trypsin inhibitor  followed by 1 volume of 2× SDS buffer. The samples were run on 10% SDS-PAGE gels and subjected to immunoblot analysis.Digestion with TPCK-trypsin was done on membranes  preparedTM XRS+ with Image LabTM software (Bio-Rad). The results were expressed as an average of triplicate experiments ± S.D. The Student's two-tailed t test was used to determine statistical significance (p < 0.001).The signals from the immunoblots were visualized and quantified using the ChemiDocA. The 1280 amino acids are organized into two nucleotide-binding domains (NBDs) and two TM and TMD. Each TMD contains 6 TM segments that extend into the cytoplasm to form ICLs that connect the NBDs to the TMDs. The TMDs interlink  to form two divergent wings that attach to NBD1 or NBD2. The protein alternates between open and closed conformations during the predicted catalytic cycle . Sites 1 and 2 are in one wing (attached to NBD2), whereas site 3 is in the lipid layer between the TM segments of both wings. One approach that could be used to identify domain interfaces affected by substrate binding is cysteine cross-linking protection assays. Cross-linking of cysteines can be readily detected because a disulfide bond between different domains causes the protein to migrate slower on SDS-PAGE gels . Therefore binding of tariquidar at the interface would be predicted to block cysteine cross-linking.The predicted tariquidar-binding sites all lie at interfaces between domains of the two homologous halves of P-gp. Site 1 is at the interface between ICL2  and ICL3 , whereas sites 2 and 3 lie at the interface between TM segments in TMD1 and TMD2. Sites 1 and 2 are on one structural wing, whereas site 3 is located at the interface between the two wings B. TherefA). Mutants containing cross-linkable cysteines between NBD1 and NBD2 (C431(NBD1)/L1176C(NBD2)  , L531C(N6C(NBD2) , and P516C(NBD2)  were inc , L531C/R741C(TM7), I299C(TM5)/F770C(TM8), T333C(TM6/L975C(TM12)) (A). By contrast, a higher concentration (1000 nm) of tariquidar did not significantly inhibit cross-linking of the ICL (L175C(ICL1)/N820C(ICL3), A259C(ICL2)/W803C(ICL3), A266C(ICL2/F1086C(NBD2)) (B) or the NBD (C431(NBD1)/L1176C(NBD2), L531C(NBD1)/C1074(NBD2), and P517C(NBD1)/I1050C(NBD2)) mutants (C). These results suggest that tariquidar likely binds to a site at the interface between TMD1 and TMD2 within the lipid bilayer (sites 2 or 3) rather than at the ICL2/ICL3 interface (site 1). This is supported by the recent observation that 500 nm tariquidar efficiently rescued 25 mutants with processing mutations in the ICL2/ICL3 interface (14 mutants in ICL2 and 11 mutants in ICL3) ) A. By conC(NBD2)) B or the  mutants C. These in ICL3) .N-glycosylated sites in the extracellular loop that connects TM segments 1 and 2 (A). Wild-type P-gp is initially synthesized as a 150-kDa core-glycosylated protein in the endoplasmic reticulum (ER). The protein then leaves the ER and the carbohydrate is modified in the Golgi to yield a 170-kDa mature protein that is then delivered to the cell surface (maturation). The presence of a processing mutation traps P-gp in the ER as a partially folded protease-sensitive core-glycosylated intermediate that is rapidly degraded. Expression of P-gp processing mutants in the presence of drug substrates promotes maturation of the protein to yield functional P-gp at the cell surface  into a lipid face destabilizes a TM segment because of the large free energy barrier of about 17 kcal/mol for exposing it to lipid hydrocarbon  and it yields the 150-kDa immature form of P-gp as the major product when expressed in the absence of drug substrates . Drug suV parent . If an am) were required to promote maturation of the 150-kDa protein to yield mature P-gp as the major product. Rescue with cyclosporine A, however, was similar to the G251V parent. By contrast, the Y307R mutation inhibited rescue with both tariquidar and cyclosporine A.Two examples of arginine mutations that inhibited rescue of G251V are shown in m tariquidar are shown in The effects of arginines introduced into the TM segments of TMD1 on rescue of G251V with 0.5 μA and E). One arginine mutation predicted to line the drug-binding pocket inhibited rescue in TM2 (A129R) (B) and two arginines predicted to line the drug-binding pocket in TM6 (F336R and F343R) were found to inhibit tariquidar rescue (F). No arginines predicted to line the drug-binding pocket were found to block tariquidar rescue in TM segments 3 or 4 (C and D).In the N-terminal TMD1 domain, the largest number of arginine mutations predicted to line the drug-binding pocket that inhibited tariquidar rescue were located in TM1  and TM5  , A and E (A129R) B and twor rescue F. No args 3 or 4 , C and DA), TM10  , TM11  , and TM12  . No arginines predicted to line the drug-binding pocket were found to block tariquidar rescue in TM segments 8 or 9 (B and C).Tariquidar rescue of the G251V/arginine mutants in TMD2 are shown in Fig. 6m cyclosporine A as this concentration showed robust rescue of the G251V parent , TM5 , TM7 (Q725R and F728R), TM10 (I868R and G872R), TM11 , and TM12 (V982R)  . The oth (V982R) . The res2R . The A potential disadvantage of the tariquidar-rescue assay is that it involves rescue of a P-gp processing mutant. Would the arginine mutations disrupt tariquidar interactions in a wild-type background? To address this question, we assayed for tariquidar-stimulated ATPase activity of purified arginine mutants. Drugs modulate P-gp ATPase activity  and the m cyclosporine A to promote maturation. All of the arginine mutants in the wild-type background yielded mature P-gp as the major product when expressed at low temperature (30 °C) in the presence of cyclosporine A (data not shown). P-gp processing mutants resemble processing mutants of its CFTR sister protein as expression at low temperature (30 °C) promotes maturation of the mutants . Wild-type P-gp ATPase activity was maximally stimulated about 8-fold (A) and had a half-maximal stimulation (S50) at a concentration of about 0.8 μm tariquidar (B). Seventeen of the 30 G251V/arginine mutants  that could not be rescued with tariquidar showed little or no stimulation of ATPase activity with tariquidar (A). Mutants L65R(TM1), A129R(TM2), F732R(TM7), Y950R(TM11), Y953R(TM11), and F978R(TM12) showed partial activity (about 25–50% of wild-type activity), whereas the maximal tariquidar-stimulated ATPase activity of mutants H61R(TM1), G64R(TM1), F303R(TM5), F343R(TM6), Q725R(TM7), V865R, and L975R(TM12) was about 50–100% of wild-type enzyme. All of the active arginine mutants, however, had S50 concentrations that were at least 5-fold higher than wild-type P-gp (B).The isolated mutant P-gps were mixed with lipid and assayed for ATPase activity in the presence of various concentrations of tariquidar. Wild-type P-gp exhibited basal ATPase activity of about 0.1 μt 8-fold A and hadriquidar B. Seventriquidar A. Mutantype P-gp B.in silico to be present in site 3 (B)The decrease in the affinities of these mutants for tariquidar in their ATPase activities was consistent with their inability to be rescued with tariquidar  and 6. Tn site 3  of the tn site 3 BRescue of processing mutants by tariquidar and cyclosporine A showed differences because many mutants containing processing mutations outside the drug-binding pocket could only be rescued with tariquidar . TariquiOne possibility is that rescue by tariquidar resembles the rescue of CFTR processing mutants with VX-809 . CorrectA and B).Therefore, we tested if tariquidar would promote maturation or stabilize P-gp TMD1. HEK 293 cells were transfected with A52-tagged TMD1 (residues 1–379), A52-tagged TMD2 (residues 681–1025), or A52-tagged TMD1 plus A52-tagged TMD2 and grown in the absence or presence of tariquidar. Whole cell SDS extracts were then subjected to immunoblot analysis . Maturatf) and F (PNGase F). Samples were then subjected to immunoblot analysis. Fig. 10C shows that tariquidar promoted maturation of TMD1 such that the mature product was resistant to endoglycosidase H but not to endoglycosidase F. By contrast, the immature form of TMD1 was sensitive to both endoglycosidases. These results indicate that the immature product is in the ER, whereas the mature form of TMD1 has traversed the Golgi apparatus.A52-tagged TMD1 together with untagged TMD2 were co-expressed in HEK 293 cells in the presence of tariquidar. The cells were solubilized and treated with endoglycosidases H (HD and E). About 10 μg/ml of trypsin was required to reduce the amount of immature TMD1 by 50%, whereas mature TMD1 remained resistant to trypsin at the highest concentration tested (1000 μg/ml) (E).We previously showed that P-gp folds into a more compact conformation when it leaves the ER such that it becomes more resistant to protease digestion . Therefo0 μg/ml) E.The results showed that tariquidar differed from cyclosporine A. We previously reported that cyclosporine A did not stabilize TMD1 . Here weA) in the predicted drug-binding pocket where arginines caused major disruptions in the ability of tariquidar to rescue processing mutants or activate ATPase activity. McCormick et al. (B). By contrast, only 2 of 13 residues of the predicted site 2 tariquidar-binding site showed overlap with residues identified by arginine mutagenesis (C). Therefore, the results of the cross-linking, arginine mutagenesis studies, and molecular docking predictions suggest that predicted site 3 is the likely high affinity-binding site for tariquidar.Through arginine mutagenesis we identified 30 positions A in the k et al.  used molk et al. B. By conagenesis C. TherefB). Binding of tariquidar at this site would enable it to stabilize both TMDs (enhanced corrector activity) and bring the two wings and NBDs together into a closed conformation that would result in stimulation of ATPase activity but with no efflux activity (inhibitor). This would be consistent with the observation that tariquidar appears to trap P-gp in a closed conformation (Binding of tariquidar to site 3 might help explain the mechanism of its effectiveness as a P-gp corrector and inhibitor. Site 3 is located in the lipid bilayer at the interface between the two wings of P-gp B. Bindinormation .A) that were not predicted to lie within 4.5-Å of the predicted site 3 tariquidar-binding site , Gln946 (TM11), and Leu975 (TM12)) that were in agreement with our results. Because there is no high-resolution structure of human P-gp, the different residues identified to be involved in tariquidar binding by the two in silico methods could be due to different algorithms and programs used to generate the homology models of human P-gp for the docking studies. It could also be due to the scoring function chosen for best docking  A that we docking . The low docking .in silico docking studies was that some arginines might have perturbed the structure of TM segments. For example, 8 of the 13 arginines with reduced tariquidar interactions are located in TM1 and TM11 (A). Cysteine cross-linking studies showed that TM segments 1 and 11 in human P-gp were adjacent to each other and that interactions between TM segments 1 and 11 were altered during the catalytic cycle by ATP hydrolysis  of a drug substrate/inhibitor by biochemical methods. The results in this study suggest that tariquidar binds to a binding site that is within the TMDs and is consistent with the idea that there is a large flexible drug-binding pocket with the TMDs that can accommodate other drug substrates  of tariquidar to activate ATPase activity by 50% (m) because the presence of detergent increases the apparent affinity of drug substrates for P-gp by 100-fold or more  and CFTR (VX-809) stabilized the TMD1 domain. Our tariquidar mapping studies suggest that molecular docking studies can be a useful approach to identify a target site.Both authors designed and conducted the experiments. Both authors analyzed the data, wrote the article, and approve of the version to be published.


Current influenza control strategies require an active surveillance system. Pseudotyped viral particles (pp) together with the evaluation of pre-existing immunity in a population might satisfy this requirement. However, the reliability of using pp in neutralizing antibody (nAb) detection are undefined.2 value equal to or near 1 in two different populations. In the quantitative detection on nAbs, although the geometric mean titers  differed between the pp and viruses, no significant difference was observed. Furthermore, humoral immunity against the reassortants was evaluated; our results indicated strong consistency between the nAbs against reassortant pp and those against naïve pp harboring the same hemagglutinin.Pseudotyped particles of A(H1N1)pmd09  and HPAI H5N1 (A/Anhui/1/2005), as well as their reassortants, were generated. The reliability of using these pp in nAb detection were compared concurrently with the corresponding viruses by a hemagglutination inhibition test, as well as ELISA-, cytopathic effect-, and fluorescence-based microneutralization assays. In the qualitative detection on nAbs, the pp and their corresponding viruses were in complete agreement, with an RThe pp displayed high reliability in influenza virus nAb detection. The use of reassortant pp is a safe and convenient strategy for characterizing emerging influenza viruses and surveying the disease burden. Influenza viruses have caused flu pandemics multiple times throughout history. There have been four major flu pandemics since 1918. The 1918–1919 pandemic H1N1 virus infected approximately 20–40% of the world's population and led to an estimated death toll of 50 million people, while the 1957–1958 pandemic H2N2 virus originated in Asia and led to 1–1.5 million deaths. Similarly, the 1968–1969 pandemic H3N2 virus killed an estimated 1 million people worldwide. Most recently, the 2009 pandemic H1N1 influenza virus resulted in an estimated 151,700–575,400 deaths worldwide during its first year of circulation Along with advancements in biomedical technology and collaboration among international organizations and national governments, the responses to previous communicable disease pandemics have resulted in the following standard procedures: disease surveillance, pathogen identification, epidemic situational reporting and surveillance, public health interventions when necessary, and vaccine and drug development Pre-existing immunity is an important factor that affects pandemic trends and limits the pandemic scale of communicable diseases Current major approaches to the evaluation of humoral immunity include the hemagglutination inhibition test and ELISA-, cytopathic effect (CPE)-, and fluorescence-based microneutralization assays. However, these methods rely on virus strain isolation and amplification, which present a challenge when dealing with highly pathogenic influenza viruses because many institutes or laboratories lack strict biological safety facilities. On the other hand, virus reassortment presents ethical issues because artificial viruses could cause a fatal pandemic. Therefore, a safe and convenient substitute model system is a precondition for active defense against influenza virus pandemics.Pseudotyped viral particles (pp) are used widely as a reliable substitute for viruses that are difficult to isolate and culture in molecular biological studies of viral envelope proteins, viral receptor exploration, and neutralizing antibody (nAb) assays. Such studies have demonstrated the availability and utility of pp. For a long time, virologists ignored this technology because influenza viruses are easy to obtain. However, pseudotyped influenza viral particles offer the following advantages: there is no restriction due to biological safety, and reassortment based on hemagglutinin (HA) and neuraminidase (NA) is easy and without ethical issues. Thus, pseudotyped influenza viral particles combined with nAb assays might be optimized to create a surveillance system for assessing pandemic trends and the related disease burden.In this study, a series of comparative analyses between live viruses and related pp were performed using the hemagglutination inhibition test and ELISA-, CPE-, and fluorescence-based microneutralization assays. Our findings provided a convenient and sensitive approach to the accurate assessment of virus-specific nAbs, which would improve pandemic influenza control and prevention.This study was conducted according to the principles of the Declaration of Helsinki. The study was approved by the Ethics Committee of Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, permit number: [2013]1. All donors gave written informed consent for research use of blood samples. We also obtained written informed consent from the guardians on the behalf of the minors enrolled in our study .This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal study was also approved by the Committee on the Ethics of Animal Experiments of Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College (Permit Number: [2013]3).The HPAI virus, A/Anhui/1/2005 (H5N1), belonging to the 2.3.4 clade, was originally isolated from a fatal Chinese case cDNA fragments encoding the full-length HA of A/California/05/2009 (GenBank Acc. No. FJ966952) and the full-length NA of A/Ohio/07/2009 (GenBank Acc. No. FJ969534) were synthesized and inserted into an expression vector pCMV  to produce constructs 09H1 and 09N1 as previously described 6 293T cells cultured in a 9.6-cm dish were co-transfected with 0.8 µg of MLV-gag-pol, 0.8 µg of CMV-GFP, 0.3 µg of pCMV-HA and 0.3 µg of pCMV-NA by using Polyfect Transfection Reagent from QIAGEN . After 6–8 h incubation, cells were washed once with phosphate-buffered saline and cultured in 3 mL complete Dulbecco minimum essential medium . At 48–72 h post-transfection, the pp were harvested from the supernatants of the transfected cells by filtration through a 0.45-µm Durapore PVDF membrane filter .The pp were produced by transfecting 293T cells with four plasmids: an HA expression plasmid pCMV-HA, an NA expression plasmid pCMV-NA, a gag-pol-encoding plasmid MLV-gag-pol, and a reporter plasmid CMV-GFP. HA and NA of naïve pp were derived from the same virus. For reassortant pp, HA and NA were from different viruses, including H5N1(09)pp, in which HA was AH H5 and NA was 09N1, and H1N1(H5)pp, in which HA was 09H1 and NA was AH N1. We carried out the following procedures: 1.5×10MDCK, A549 targeting cells and 293T human embryo kidney pseudoparticle producer cells were obtained from the American Type Culture Collection  and grown in Dulbecco's modified essential medium  supplemented with or without 10% fetal bovine serum.The pp or virus samples were serially diluted 1∶2 in phosphate-buffered saline (PBS) in microplate wells (50 µL/well). Next, 50 µL aliquots of turkey red blood cells (1% suspension) were added to each well. Hemagglutination was scored 30 min later. Hemagglutination inhibition tests were performed according to standard methods 50)/well were incubated with two-fold serial dilutions of RDE-treated sera or NIBSC standard substance in a 96-well plate at 37°C in the presence or absence of TPCK-trypsin for 2 h. Then, 100 µL of freshly trypsinized MDCK cells (1.5×105/mL) were added to each well . After 18–22 h of incubation at 37°C and 5% CO2, the monolayers were washed with PBS and fixed in cold 80% acetone for 10 min at room temperature, then air-dried. The presence of viral protein was detected by ELISAs using a monoclonal antibody against the influenza A NP (clone F8) or HA, that is, NP for viruses and HA for pp. Anti-NP or -HA antibodies diluted 1∶4,000 in PBS containing 1% bovine serum albumin and 0.1% Tween 20 (E diluent) were added to each well and incubated at room temperature for 1 h. The plates were then washed and horseradish peroxidase-conjugated anti-mouse IgG  diluted 1∶2,000 in E diluent was added to each well, followed by further incubation for 1 h. After washing, o-phenylenediamine dihydrochloride was added and incubated at room temperature for 5 min. The reaction was stopped with the addition of an equal volume of 1 N sulfuric acid. The absorbance at 490 nm was then measured. The endpoint titer was expressed as the reciprocal of the highest dilution of serum with an OD value less than X, where X =  [(average of V+C wells) − (average of C wells)]/2+ (average of C wells) Viruses or pp with 100 50% tissue culture infectious dose  or 37°C (H5N1 and pp) in the presence or absence of TPCK-trypsin for 1 h. The mixtures were then transferred to MDCK cell monolayers in a 96-well plate and incubated at 35 or 37°C for 1 h. After washing once, the cells were cultured in medium with TPCK-trypsin for virus or without for pp to prevent viral re-entry. The plates were then incubated at 35 or 37°C with 5% CO2 for 48–72 h. After this incubation period, the formation of a CPE or fluorescence was observed using a Nikon fluorescence microscope . Interpretation of the results was performed by comparing positive virus controls and negative cell controls. The nAb titer was defined as the highest dilution of serum or antibody at which 95% infectivity was blocked.Viruses or pp with 100 TCIDIn total, 270 serum samples were obtained from in- and out-patient subjects  in a cooperative hospital in Shanghai, China, in 2006. The subjects were considered to have not been exposed to A(H1N1)pmd09 influenza virus. An additional 286 samples were taken from another cooperative hospital in Kunming, China, in 2013. All study subjects were selected on the basis of a questionnaire designed to exclude any person with symptoms possibly associated with an influenza-like illness in the previous 6 months, or with an influenza virus vaccination history, especially the 2009 H1N1 vaccination for the 2013 subjects.NIBSC antiserum 07/338, Influenza antiserum A/Anhui/1/05 (H5), NIBSC code 07/338; NIBSC antiserum 07/150, International Standard for antibody to A/Vietnam/2004 H5N1 Clade 1 virus, NIBSC code 07/150, and NIBSC antiserum 09/152, influenza anti-A/California/7/2009 (H1N1)v HA serum, NIBSC code 09/152, were purchased from the NIBSC.Monoclonal antibodies were purchased from Biovisualab Inc. . Goat anti-influenza virus sera were raised by immunizing goats with purified H5N1delta or NYMC X-179A in our laboratory.The supernatant of the medium containing pp was concentrated at 40,000 rpm for 25 min in a Hitachi centrifuge . Subsequently, both the original and concentrated supernatants were negative-stained, and the pp were observed by transmission electron microscopy  using an acceleration voltage of 80 kV.t-test, and estimation of geometric mean titers (GMTs) with confidence intervals and corresponding P-values, using SAS software (ver. 9.1). All data were expressed as the mean ± standard deviation or GMT .To estimate the value of the MN titer corresponding to the ELISA titer of anti-2009 and -2005 HA antibodies, we performed a correlation analysis using linear regression models. Data were analyzed using linear regression and multivariable models, Student's To evaluate the reliability of using pp as a substitute for the relevant influenza virus in nAb analysis, we first compared pp of A(H1N1)pmd09  and HPAI H5N1 (A/Anhui/1/2005) with their corresponding attenuated virus (NYMC X-179A and H5N1delta) in a hemagglutination inhibition test and ELISA-, CPE-, and fluorescence-based microneutralization assays. Goat antisera immunized with NYMC X-179A and H5N1delta and one anti-A(H1N1)pmd09 monoclonal nAb were used in these parallel tests. To ensure standardization and comparability, two NIBSC antisera against H5, 07/338 [A/Anhui/1/05 (H5)] and 07/150 [A/Vietnam/2004 H5N1 clade 1 virus], and one NIBSC antiserum against A/California/7/2009 (H1N1)v (09/152) were also included in these tests.In the hemagglutination inhibition test, the viruses and pp were adjusted to 4 hemagglutination units according to WHO guidelines. As listed in 50 according to WHO guidelines. The highest dilution titer of NIBSC antiserum 07/338, NIBSC antiserum 07/150, and goat immune serum H5 to neutralize 100 TCID50 H5N1delta and H5N1pp were 1067±230.94 and 1000±346.41, 0.13±0.02 and 0.14±0.02 IU, and 4000±0.00 and 5000±1732.05, respectively. The highest dilution titer of anti-H1 monoclonal antibody, NIBSC antiserum 09/152, and goat immune serum H1 to neutralize 100 TCID50 NYMC X-179A and H1N1pp were 0.83±0.00 and 0.69±0.12 pmol, 2400±0.00 and 2933±461.88, and 3000±0.00 and 4667±1154.7, respectively pdm09 pandemic. Comparative parallel tests were performed using these sera first.NYMC X-179A and the H1N1pp detected a similar number of subjects with nAbs against A(H1N1)pmd09. The titers against NYMC X-179A and H1N1pp as measured by a hemagglutination inhibition test and by ELISA-, CPE-, and fluorescence-based microneutralization assays were (GMT [95% CI]) 25.78  and 34.79 , 24.67  and 24.17 , and 19.48  and 21.58 , respectively .In comparison, H5N1delta and the H5N1pp detected a similar number of subjects with nAbs against H5N1. The titers against H5N1delta and H5N1pp as detected by a hemagglutination inhibition test and by ELISA-, CPE-, and fluorescence-based microneutralization assays were 17.82  and 14.86 , 12.01  and 10.7 , and 11.07  and 10.7 , respectively .2 value equal to or near 1. By quantitative detection, although the H1N1pp were more sensitive than NYMC X-179A, the pseudotyped particles showed fairly high consistency with the corresponding virus.In conclusion, by qualitative detection, the pseudotyped particles showed complete agreement with the corresponding virus, with an RThe second set of sera, collected from 270 subjects in 2006, represented the population immunity characteristics before the A(H1N1)pdm09 pandemic. To further evaluate the reliability of using pp in nAb detection, comparative parallel tests were performed using these sera.NYMC X-179A and the H1N1pp detected the same 68 of 270 serum samples carrying nAbs against A(H1N1)pdm09. The titers against NYMC X-179A and the H1N1pp as detected by a hemagglutination inhibition test and by ELISA-, CPE-, and fluorescence-based microneutralization assays were 33.54  and 30.54 , 63.45  and 63.58 , and 56.42  and 59.32 , respectively .In comparison, H5N1delta and the H5N1pp detected the same 5 of 270 serum samples with nAbs against H5N1. The titers against H5N1delta and the H5N1pp as detected by a hemagglutination inhibition test and by ELISA-, CPE-, and fluorescence-based microneutralization assays were 20.41  and 19.36 , 21.10  and 17.68 , and 17.83  and 19.14 , respectively .In conclusion, using both qualitative and quantitative detection methods, the pseudotyped particles showed fairly high consistency with the corresponding virus. Furthermore, the immune protection level of the population against A(H1N1)pdm09 was high.The influenza virus pandemic surveillance system described in this report included two major elements: 1) predictions of HA and NA matching with reassortant pp, and 2) evaluation of the background protection level of population immunity against a new reassortant using valid reassortant pp.Toward realizing this goal, we produced reassortant pp, including H5N1(09)pp and H1N1(H5)pp. To study the population immunity protection level, the above two reassortant pp as well as naïve pp (H1N1pp and H5N1pp) were used to perform hemagglutination inhibition testing and CPE- and fluorescence-based microneutralization assays on the second group of serum samples.The H5N1pp and H5N1(09)pp detected the same 5 of 270 serum samples containing nAbs against H5. The anti-H5N1 and -H5N1(09)pp titers (GMT [95% CI]) in the hemagglutination inhibition test and CPE- and fluorescence-based microneutralization assay were 19.36  and 18.35 , and 19.14  and 16.57 , respectively .In comparison, the H1N1pp and H1N1(H5)pp detected the same 68 of 270 serum samples with nAbs against H1. The anti-H1N1pp and -H1N1(H5)pp titers in the hemagglutination inhibition test and CPE-based microneutralization assay were 30.54  and 41.35 , and 59.32  and 58.54 , respectively .2 = 1). The average nAb titers against reassortant pp were slightly lower than that against the native pp. These findings suggested that pseudotyped influenza viral particles combined with an nAb assay might be optimized to constitute a surveillance system for pandemic trends and the related disease burden.In summary, both the naïve and reassortant pp displayed complete agreement with each other using qualitative detection methods ; and envelope proteins, which are presented on the artificial viral particle pmd09  and HPAI H5N1 (A/Anhui/1/2005). Third, the reliability was verified using two population groups, representing the population immunity situation before and after the A(H1N1)pmd09 outbreak; the nAb-positive rate of the latter was much higher than that of the former. After we demonstrated the reliability of using pseudotyped influenza viral particles in nAb detection, we generated reassortant pp using the NA and HA of A(H1N1)pmd09 and HPAI H5N1 (A/Anhui/1/2005) to examine the possibility of pp in emerging influenza disease burden estimation. For pp harboring either H5 [H5N1pp and H5N1(09)pp] or H1 [H1N1pp and H1N1(H5)pp], strong consistency was detected in both quantitative and qualitative nAb analyses.The host immune barrier might be caused by cross-infection or an inapparent infection, and all pathogens have their own evolutionary process. For example, despite the initial concern that little protective immunity against the novel H7N9 virus existed in the general population, subsequent epidemiological studies suggested the occurrence of pre-existing immunity Both population groups displayed very low nAb-positive rates against H5N1 virus and pp harboring H5, suggesting that the immunity level to this virus was very low. Of the nAbs against A(H1N1)pmd09 and pp harboring the same HA, the rate of positive sera sampled in 2006 was less than that of the sera sampled in 2013. This suggested that after the A(H1N1)pmd09 pandemic, populations were immunized broadly to a certain extent. The nAb titers of the sera sampled in 2006 were slightly higher than those of the sera sampled in 2013. This might be due to the difference in population. The sera sampled in 2006 demonstrated high pre-existing immunity to the A(H1N1)pmd09, which was consistent with a strong pre-existing immunity against A(H1N1)pmd09 reported in Shanghai Virological and molecular virological characterization of HA and NA expression and maturation, HA and NA incorporation into pp, the conformational and functional consistency of HA and NA between pp and the corresponding wild-type virus, pp stability and infectivity, and so on have been well characterized in our and other researchers' studies The pp were also applied for other novel influenza viruses, and a recent study developed pseudotyped viral particles to detect neutralizing antibodies against H7N9 The pp were a single cycle system and not able to capture events downstream of entry/fusion such as inhibition of egress and inhibition of HA maturation. However, these downstream inhibition steps have been described for many broadly neutralizing monoclonal antibodies and they might be important for heterosubtypic immunity. Therefore, there is a limitation of pp in that the pp system cannot represent these biological processes of corresponding virus.


We determined that the extent of ITH varied among the three cases. On average, 65% of all the mutations detected were common within individual tumors. KMT2C aberrations and the NCOR1 mutation were the only ubiquitous events. Subsequent phylogenetic analysis showed that the tumors evolved in a branched manner. Comparison of the primary and metastatic tumors revealed that PPP2R1A (E370X), SETD2 (I1608V), SMAD4 (G382T), and AR splicing site mutations may be specific to liver metastatic cancer. These mutations might contribute to the initiation and progression of distant metastasis. Collectively, our analysis identified a substantial level of genetic ITH in CRC, which should be considered for personalized therapeutic strategies.Intratumor heterogeneity (ITH) leads to an underestimation of the mutational landscape portrayed by a single needle biopsy and consequently affects treatment precision. The extent of colorectal cancer (CRC) genetic ITH is not well understood in Chinese patients. Thus, we conducted deep sequencing by using the OncoGxOne Colorectal cancer (CRC) is one of the leading causes of death worldwide, accounting for an estimated 1.2 million new cases and 600,000 deaths every year . TherefoAPC, KRAS, and TP53 mutations in all regional biopsies. The sequencing coverage of their study is relative low, which may lead to overlook the low frequency mutation. Whereas in Kogita study, [Intratumor heterogeneity (ITH) leads to an underestimation of the mutational landscape portrayed by a single needle biopsy and consequently affects treatment precision. With the development of next-generation sequencing (NGS), ITH has recently been elucidated in substantial detail in several cancer types –7, inclua study,  they onl™ Plus panel of DNA from multi-region biopsies of primary and liver metastatic tumors of three Chinese CRC patients to characterize CRC ITH in Chinese patients. We determined the mutation landscape and identified the substantial level of genetic ITH in CRC.We conducted deep coverage sequencing by using the SureSelect Target Enrichment Kit and OncoGxOneThree patients with multi-regional primary colon carcinoma surgically resected in the First People’s Hospital of Yunnan Province between October 2013 and April 2014 were enrolled in the study. Pathological tumor staging was conducted in accordance with the TNM classification . This st® DNA Mini Kit  according to the manufacturer’s procedure with slight modifications. Genomic DNA samples were quantified by using the NanoDrop 2000 spectrophotometer . The isolated DNA was stored at −80°C until analysis.The Formalin-fixed, paraffin-embedded (FFPE) specimens were subjected to histological review, only those containing sufficient tumor cells (at least 70% tumor cells), as determined by hematoxylin and eosin staining, and matched normal tissues were selected for DNA isolation. Genomic DNA was isolated by using the QIAampThe microsatellite instability (MSI) status was determined for each case by using five mononucleotide or dinucleotide microsatellites markers  . Primers™ Plus cancer panel  according to the manufacturer’s instructions. OncoGxOne Plus, a targeted oncology panel, contains 333 cancer-specific genes . Briefly, 200 ng of genomic DNA from each sample was fragmented by acoustic shearing on a Covaris instrument. Fractions of 150–200 bp were ligated to SureSelect Adaptor Oligo and PCR-amplified for 10 cycles. For target enrichment, the entire library was hybridized by using the SureSelect Oligo Capture Kit for 16 or 24 h at 65°C. Biotinylated hybrids were captured, and the enriched libraries were completed under 16 PCR cycles. The resulting purified libraries were pooled and sequenced by using the Illumina® MiSeq™ NGS instrument for 2 × 150 paired-end sequencing reads  according to the manufacturer’s protocols. The genes in the OncoGxOne™ Plus cancer panel are listed in Sample sequence enrichment and library preparation was conducted by using the SureSelect Target Enrichment Kit  and OncoGxOnehttp://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/) and then aligned to the human reference genome (hg19) by using the Burrows–Wheeler alignment tool to generate a bam file [Sequencing data were accessed through the MiSeq Reporter. Data quality was checked by using FastQC  were included in this study. Then, 10 samples from 3 patients were sequenced, with a median coverage of 250 reads. We identified ~8,000 somatic single-nucleotide variations (SNVs)  and ~850  somatic insertions and deletions (indels) in the 10 biopsy samples from the 3 CRC cases .A 62-year-old male was diagnosed with MSS colon cancer, with a 3.5 cm × 3 cm × 1.2 cm tumor. Two regions (P1-1 and P1-2) of the primary tumor exhibited a moderately differentiated adenocarcinoma, and the pathological stage of the tumor was T2N1M0. The patient received no prior treatment.APC (428_429del), KIT (A755T), KMT2C , NCOR1 (S63L), NRAS (G12D), and PIK3CG (K344Q) mutations. P1-1 contained seven additional mutations, namely, APC (Q706X), KMT2D (3860_3861del), KMT2C  BRCA2 (T3030fs), and ERBB4 splicing site mutation. P1-2 contained another AR (57_58del) mutation.The nonsynonymous somatic point mutations and indels that lead to protein alterations are summarized in A 60-year-old female was diagnosed with MSS colon cancer, with a 3.8 cm × 3.5 cm × 1.2 cm tumor. Three regions  of the primary tumor exhibited a moderately differentiated histology and right hepatic lobe metastasis. Multiple lymph node metastases (pN2) were detected, and the pathological stage of the tumor was T2N4M0. The patient received no prior treatment.For Patient 2, targeted resequencing of DNA from the three regions of primary tumor was conducted. This process revealed 25 nonsynonymous point mutations and 8 indels . Their dATRX (P571A), CHEK2 (P358S), KDM6A (A1038T), NCOR1 , KMT2C , and AR (57_59del), which were ubiquitous mutations. The TGFBR2 (E125fs) was specific to P2-1, SETD2 (I1608V) mutation was specific to P2-2, TP53 (T218P), KMT2C (K339N) was specific to P2-3. APC (L693fs) and TP53 (R89W) was shared by P2-2 and P2-3. NCOR1 (K85N) was shared by P2-2 and P2-1. KMT2C (Q755X) is shared by P2-1 and P2-3.We identified several significantly mutated genes, including A 71-year-old female was diagnosed with MSS-high and moderately differentiated colon cancer, with a 9 cm × 5 cm × 3 cm primary tumor and a 1.5 cm × 1 cm × 0.5 cm right hepatic lobe metastasis. Three regions  of the primary cancer and two metastatic regions were resected, and the pathological stage of the tumor was T3N2M2. The patient received no prior treatment. For this patient, targeted resequencing was conducted on the DNA from the three regions  of the primary tumor and two metastatic regions. The process revealed 47 nonsynonymous point mutations and 11 indels . Their dWe constructed a phylogenetic tree , which sOverall, target sequencing identified 45 somatic mutations per biopsy, accounting for 78% of all mutations observed in this tumor. 60% of all the mutations detected by multi-region sequencing in the colectomy specimen were ubiquitous mutations present in all regions. When we analyzed the primary cancer, an average of 45 somatic mutations were detected per biopsy, which accounted for 87% of all the mutations identified in this tumor. For the metastatic sites, a single biopsy revealed 92% of all the mutations detected in this tumor.KRAS (G13D), PIK3CA (E545), TP53 a splicing site mutation, APC (E854fs), KMT2C , SETBP1 (Q1558L), and NCOR1 (S63L) were ubiquitous mutations, whereas CDKN2A (D74A), SMAD4 (W168X), NCOR1 (N208K), KMT2C (G315C and K306fs) and INHBA (V58I) were specific to the primary cancer. PPP2R1A (E370X), SETD2 (I1608V), SMAD4 (G382T), and AR splicing site mutations were specific to liver metastatic cancer. However, these mutations were absent in the primary cancer regions. The KRAS, TP53, and PIK3CA mutations, which have often been used as molecular biomarkers in CRC, also presented in all the regions.In the significantly mutated genes, we determined that PPP2R1A gene encodes the structural subunit of the PP2A enzyme, which is a highly conserved serine/threonine phosphatase that serves a broad spectrum of biological roles, including the negative regulation of signal transduction, cell cycle progression [By comparing the mutation landscape between primary and metastatic regions, we detected that several mutations were specific to the metastatic regions. SMAD4 is a key transducer of transforming growth factor-β superfamily signaling, which regulates cell proliferation, differentiation, and apoptosis . Loss ofgression , and gengression .The multi-region mutation landscape analysis of the three CRC tumors provided substantial evidence of ITH, with the extent of ITH varying among the three cases. In Patient 1, we sequenced two spatially separated sites of primary cancer. Their mutational spectra exhibited a 63% overlap. In Patient 2, a single biopsy exhibited 28 somatic mutations on average, accounting for 85% of all the mutations observed in this tumor. By contrast, in Patient 3, 78% of all the mutations were observed in this tumor. Of all the somatic mutations revealed by multi-region sequencing, 30% to 40% were heterogeneous and thus cannot be detected in every sequenced region. Kim et al. [We constructed a phylogenetic tree by using the data from Patient 3. The tree indicated that CRC evolved in a branched, rather than a linear, manner. The branched evolution model has been validated in many types of cancers , includiKMT2C and NCOR1 (S63L) were mutated in all regions compared with the significantly mutated genes reported by Kandoth et al. [KMT2C is frequently altered in many types of cancer, including liver cancer [KMT2C, the clinical significance of which remains to be validated. We also observed a NCOR1 missense mutation. NCOR1 is a 270 k nuclear receptor corepressor [KMT2C and NCOR1 may represent two potential targetable genes in the setting of heterogeneity.Branched tumor evolution underlines the necessity to target mutations located in the main trunk of the phylogenetic tree. We determined that h et al. . KMT2C ih et al.  and is ir cancer , cutaneor cancer , and esor cancer . In our epressor  that parepressor . KMT2C aKRAS, TP53, and PIK3CA are the most significantly mutated genes in CRC [s in CRC . Howevers in CRC . The priPPP2R1A (E370X), SETD2 (I1608V), SMAD4 (G382T), and AR splicing site mutations were determined to be potential metastatic tumor-specific mutations. We suppose that these mutations could drive CRC metastasis. Given the limited sample size, the frequency of these mutations in CRC metastases must be assessed in a larger cohort. Further functional studies are necessary to address their roles in CRC metastasis.Genomic analysis from single needle biopsies may underestimate the mutational landscape of heterogeneous tumors. ITH may partly explain the poor validation of CRC biomarkers due to sampling bias. Reconstructing the tumor clonal structure and identifying the ubiquitous alterations located in the trunk of the phylogenetic tree may contribute to the discovery of more effective biomarkers and therapeutic approaches.In conclusion, we identified the substantial extent of ITH in CRC. In the context of this phenomenon, we determined that CRC evolved in a branched manner and several molecular events may contribute to CRC progression. We investigated only three cases because of financial constraints. Further studies with large sample sizes should be conducted to confirm these findings.S1 Table(PDF)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


E. coli (EHEC) serogroup O145 is regarded as one of the major EHEC serogroups involved in severe infections in humans. EHEC O145 encompasses motile and non-motile strains of serotypes O145:H25 and O145:H28. Sequencing the fliC-genes associated with the flagellar antigens H25 and H28 revealed the genetic diversity of the fliCH25 and fliCH28 gene sequences in E. coli. Based on allele discrimination of these fliC-genes real-time PCR tests were designed for identification of EHEC O145:H25 and O145:H28. The fliCH25 genes present in O145:H25 were found to be very similar to those present in E. coli serogroups O2, O100, O165, O172 and O177 pointing to their common evolution but were different from fliCH25 genes of a multiple number of other E. coli serotypes. In a similar way, EHEC O145:H28 harbor a characteristic fliCH28 allele which, apart from EHEC O145:H28, was only found in enteropathogenic (EPEC) O28:H28 strains that shared some common traits with EHEC O145:H28. The real time PCR-assays targeting these fliCH25[O145] and fliCH28[O145] alleles allow better characterization of EHEC O145:H25 and EHEC O145:H28. Evaluation of these PCR assays in spiked ready-to eat salad samples resulted in specific detection of both types of EHEC O145 strains even when low spiking levels of 1–10 cfu/g were used. Furthermore these PCR assays allowed identification of non-motile E. coli strains which are serologically not typable for their H-antigens. The combined use of O-antigen genotyping (O145wzy) and detection of the respective fliCH25[O145] and fliCH28[O145] allele types contributes to improve identification and molecular serotyping of E. coli O145 isolates.Enterohemorrhagic  Escherichia coli ) and O145-specific fliCH28 (fliCH28[O145]) genes (ihp1 gene of EHEC O145:H28 was described previously  gene PCR (The gene PCR .fliCH25[O145] PCR showed cross-reactions with some non-O145:H25 strains we determined the fliCH25 sequence of each one representative strain per serogroup O2:H25 (CB13990), O100:H25 (CB9767), O165:H25 (CB12546), O172:H25 (CB10528) O177:H25 (CB14727). The flagellin encoding gene derived from these strains had each a length of 1332 bp (443 aa). Homology between the fliCH25 genes of E. coli O2, O100, O145, O165, O172 and O177 was found in the target region of the fliCH25[O145] real-time PCR  and CB11499 (O103:H25). A cluster analysis of the fliCH25 coding sequences is presented in fliCH25[O145] gene PCR reacting strains . The fliCH25 sequences of PCR non-reacting strains N234 and CB11499 were found genetically more distant . The fliC sequence of the O100:H25 strain showed one nucleotide exchange at another position . These nucleotide exchanges had no effect on the derived amino-acid (aa) sequence (fliCH25[O145] PCR assay was further tested on a panel of strains including reference strains encompassing H-types H1 to H56. None of these strains gave a positive result with the fliCH25[O145] PCR (data not shown).In detail, the sequence . On the sequence . The spefliCH28[O145] sequence was tested on 70 strains identified as H28 positive by serotyping or nucleotide sequence typing of fliC genes and by a generic fliCH28 PCR targeting common regions of the fliCH28 genes. The strains belonged to 14 different O groups, as well as to ONT and Orough strains. The results are summarized in fliCH28[O145] PCR reacted with all 35 O145:H28 and two Orough:H28 strains which were positive for the O145wzy gene. Except for four O28:H28 strains, no other strain expressing flagellar type H28 reacted in the fliCH28[O145] PCR. In order to explore the nature of the observed reaction with O28:H28 strains we determined the nucleotide sequence of the fliC gene of a representative strain . The sequence had the same length and was identical to that found with O145:H28 strains.The real-time PCR developed from the fliCH28 sequences derived from fliCH28[O145] PCR reacting (E. coli O145:H28 and O28:H28) and non-reacting (E. coli O132:H28 and O152:H28) strains revealed two genetic clusters of strains that were distinguishable by the fliCH28[O145] PCR (E. coli H-type reference strains H1-H56 reacted in the fliCH28[O145] PCR except for strain 5306–56 (O26:H46) which gave a positive reaction with a cycle threshold (CT) value of 21.3. The reaction was supposed to be fliC specific as another H46 strain from our collection  reacted equally well  in the PCR. By comparing the corresponding regions in the EHEC fliCH28[O145] genes and the fliCH46 gene of strain 5306–56 , only small differences were found (one mismatch in the backward primer and one mismatch in the probe sequence). The sequence similarity in the target region could explain the PCR cross-reaction with flagellar type H46 strains. Comparison of the fliC gene sequences of five E. coli H28 strains belonging to serogroups O145 (CB12513 and RM12581), O28 (CB9651), O132 (N87) and O152 (SE11) with that of strain 5306–56 (O26:H46) revealed that the latter is genetically more distant from all these  were spiked with high (100–1000 cfu/g) and low (1–10 cfu/g) quantities of the E. coli strains described in stx1, stx2, eae, O145ihp1, O145wzy, fliCH25[O145] and fliCH28[O145]. The results are summarized in wzy, fliCH25[O145] and fliCH28[O145] detection systems described in this work.Two random 25-g portions of the salad used for spiking experiments were analyzed for their bacterial microflora. The total mesophilic counts on Standard I agar (25°C) were 2.06–3.26 x 10E. coli O145 are known as the causative agents of severe illness such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) worldwide. EHEC O145 encompass motile and non-motile strains of serotypes O145:H25 and O145:H28. While EHEC O145:H28 strains have been described for many years, EHEC O145:H25 were identified more recently as a second important type among serogroup EHEC O145 strains and were isolated from human patients [Shiga-toxin producing patients , 39–40.ihp1 gene [ihp1 PCR-assay does not react with O145 strains carrying other H-antigens [E. coli O145 O-antigen specific wzx or wzy sequences as targets for specific detection of these strains . Many of the so far described real-time PCR assays use combinations of different genes for a more specific detection of EHEC O145 and other EHEC strains. For example, assays targeting both eae-γ1 (intimin) and fliCH28 sequences were developed for specific detection of EHEC O145:H28 strains [fliCH25 and the eae-β genes [A number of different real-time PCR assays were developed for detecting EHEC O145 strains. Some of these methods are based on the presence of the hp1 gene , 41 whichp1 gene . Howeverantigens  includinstrains  E. coli strains which are serologically not-typable for their H-antigens [In this study we have employed the sequence diversity of antigens , 21–22. FliC genotyping was also found useful to discriminate clearly between serologically cross-reacting flagellar antigens H8 and H40 and genetic subtypes of flagellar antigen H8 were identified and associated with EHEC O111 and STEC strains belonging to other O-groups [fliC genes encoding the H7 antigen in E. coli strains belonging to different O-groups revealed ten different alleles and PCRs specific for the fliC gene carried by EHEC O157:H7 and O157:HNM were developed for DNA-based typing [O-groups . Analysid typing –25.eae-gene [E. coli strains belonging to different O-groups and pathotypes  prevents the design of a real-time PCR assay that would allow a complete discrimination.In order to better characterize EHEC O145:H25 and EHEC O145:H28 strains we have developed real time PCR-assays targeting the fliCH28 allele was developed. Apart from EHEC O145:H28 this variant was only found in EPEC O28:H28 strains which shared some common traits with EHEC O145:H28  [stx-positive strains.In a similar way, a specific real-time PCR assay for detection of O145:H28 targeting its SP_O145) , 45–46. 6 CFU/g salad) did not affect detection of the inoculated EHEC strains.Evaluation of the specific real-time PCR assays for EHEC O145:H25 and O145:H28 strains with artificially contaminated ready-to eat salad samples resulted in specific detection of both types of EHEC O145 strains even when inoculated in low quantities (1–10 CFU/g) after overnight enrichment. The presence of a dense natural microflora Click here for additional data file.


MECP2 gene, which encodes a chromosomal protein that binds to methylated DNA. Mouse models mirror the human disorder and therefore allow investigation of phenotypes at a molecular level. We describe an Mecp2 allelic series representing the three most common missense Rett syndrome (RTT) mutations, including first reports of Mecp2[R133C] and Mecp2[T158M] knock-in mice, in addition to Mecp2[R306C] mutant mice. Together these three alleles comprise ∼25% of all RTT mutations in humans, but they vary significantly in average severity. This spectrum is mimicked in the mouse models; R133C being least severe, T158M most severe and R306C of intermediate severity. Both R133C and T158M mutations cause compound phenotypes at the molecular level, combining compromised DNA binding with reduced stability, the destabilizing effect of T158M being more severe. Our findings contradict the hypothesis that the R133C mutation exclusively abolishes binding to hydroxymethylated DNA, as interactions with DNA containing methyl-CG, methyl-CA and hydroxymethyl-CA are all reduced in vivo. We find that MeCP2[T158M] is significantly less stable than MeCP2[R133C], which may account for the divergent clinical impact of the mutations. Overall, this allelic series recapitulates human RTT severity, reveals compound molecular aetiologies and provides a valuable resource in the search for personalized therapeutic interventions.Rett syndrome is caused by mutations in the X-linked  MECP2 gene are implicated in several human disorders characterized by developmental delay and intellectual disability, including Rett syndrome (RTT) (MECP2 duplication syndrome (Mutations in the X-linked me (RTT)  and MECPsyndrome ,4, but ksyndrome ,6 offer syndrome . Importain vitro studies suggest that both can adversely affect DNA binding , followed by R306C ∼9%) and R133C (∼5%) (% and R13 binding –12. The  binding . T158M iMecp2 locus as fusions with the reporter protein enhanced green fluorescent protein (EGFP), including wild type (WT). We find that these RTT mutations recapitulate the severity spectrum of human RTT, and they offer a molecular explanation for this phenotypic range. In particular, the R133C mutant protein has a reduced affinity for all known MeCP2 target sequences, including methyl-CG (mCG) and is significantly reduced in abundance. We conclude that the RTT-like phenotype cannot be exclusively attributable to loss of hmC binding by this mutant protein as previously proposed  . Mice wiproposed . The greproposed . R306C, Mecp2 or mutant Mecp2 genes were fused in frame with EGFP at their C-termini . Mice expressing knock-in WT MeCP2-EGFP fusion genes have been reported (Mecp2-GFP gene (WT-GFP) were expressed in brain, though at somewhat higher levels than the endogenous Mecp2 gene (WT) . Each of the mutants gave rise to males that exhibited overt phenotypic defects from ∼6 weeks of age (see below), but their survival profiles were very different . R133C-GFP males were most mildly affected, with a median survival of 42 weeks.Using the same knock-in technology, we generated the following mouse lines: analyses  . In this respect, also the phenotypes of these frequent RTT mutations in mice reflect the spectrum of human severity.Rett syndrome affects females who are heterozygous for these T158M-GFP, R306C-GFP and R133C-GFP mutations provides validation that the mouse models are an appropriate system to investigate the mechanism by which these mutations cause RTT in humans. We used cell biological and biochemical techniques to understand the reasons for compromised MeCP2 function. At the level of mRNA, expression of the Mecp2-GFP knock-in alleles in brain was somewhat elevated compared with the native Mecp2 gene, except in the case of R133C-GFP  and fluorescence-activated cell sorting . Abundance was therefore equivalent to approximately 95 and 50% of untagged MeCP2 for R133C-GFP and T158M-GFP, respectively. Reduced stability of T158A, a distinct allele of T158, was reported previously . This relationship was also seen with an alternative WT-GFP clone (data not shown).The matching spectrum of severity between humans and mice with respect to GFP Fig. A. In agrGFP Fig. B. In coneviously . To deteSupplementary Material, Fig. S3D). This effect was more severe for T158M-GFP than for R133C-GFP. An equivalent pattern was also seen in embryonic stem cell-derived neurons in culture . The results suggest compromised binding to mCG sites by both these mutants, as explored below.Taking advantage of GFP immunofluorescence, we examined localization of the different MeCP2 variants in hippocampal sections of hemizygous male mouse brains Fig. C. The inin vivo and in vitro findings in the literature for this mutant, and they leave open the question of whether this defect has any impact on chromatin association in living cells.Published evidence regarding the DNA binding affinity of R133C protein is inconsistent. South-western ,22 and Eet al., manuscript in preparation). The method has the advantage that it detects binding of full-length protein in living cells while allowing the modification status of the target DNA to be precisely defined. In this way, we could test binding not only to mCG, but also to methyl-CA (mCA) and hydroxymethyl-CA (hmCA), which are all implicated as target sequences for MeCP2 , and therefore the effects of the mutation on protein stability as seen in mouse brain do not affect these results. Of particular note, the R133C mutation significantly compromises the interaction between MeCP2 and all modified oligonucleotides in vivo, including mCG.To address this issue, we used a cellular assay in which the various forms of MeCP2 are over-expressed transiently at equivalent levels in the presence of double-stranded oligonucleotides bearing specific modifications of cytosine  from cross-linked brain chromatin by R133C-GFP was reduced to ∼45–60% of the WT-GFP level, whereas T158M-GFP was 5–10% of WT-GFP , Mybpc3 and Krt222 (up-regulated), but only two of these were reproduced by qPCR. In one case, the effect was very small. In the other case (Abhd1), the R133C-GFP expression was unchanged relative to WT or null comparators. In fact, the variance in expression was attributable to differences between WT-GFP cerebellum and the other tested tissues . We explored the data further by binning genes according to length to replicate recent studies showing that long genes are disproportionately up-regulated in Mecp2 mutant brains . In this study, the up-regulation of long genes was modest in the Mecp2-null versus WT cerebellum, which was in agreement with previous similar analyses of this brain region ; (iii) a cellular assay showed that full-length R133C-GFP binding to mCG was greatly reduced; and (iv) ChIP analysis of genomic regions where mCG was the only detectable modification showed greatly reduced binding by the mutant protein in the brain. The brain ChIP data are particularly compelling, as mouse satellite sequences and a gene cluster, both of which lack hmC and mCH, showed impaired binding in vivo. In addition to this loss of mCG binding, transfection experiments showed a significant reduction in MeCP2[R133C] binding to the alternative canonical binding sites mCA and hmCA.Molecular and cell biological assays allowed us to critically assess a recent hypothetical explanation for the milder phenotype in ated DNA . Our finin vitro . DNA biThe introduction of specific RTT mutations into mice confirms the value of this model system in studying the molecular basis of this condition. Generalizations are starting to emerge—for example, three missense mutations  have now been found to destabilize the protein and almost certainly make a significant contribution to the phenotype (this work and reference ). It is Mecp2, Mecp2R133C, Mecp2T158M and Mecp2R306C were constructed using a 7.2-kb plasmid subclone of mouse 129/Ola genomic DNA . The coding sequence of EGFP was fused in-frame to the end of the coding sequence of Mecp2 in exon 4 followed by a loxP-flanked NeoStop cassette as a selectable marker, retaining the first 1.8 kb of the Mecp2 3′ UTR as the 3′ homology arm. Point mutations R133C, T158M and R306C were introduced into the WT Mecp2-EGFP targeting vector using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies). Linearized constructs were electroporated into 129/Ola E14 TG2a mouse ES cells and correctly targeted clones identified by PCR screening and Southern blotting . Mice were generated from ES cells by standard procedures  and then reverse transcribed  according to the manufacturer's instructions. Fifty nanograms of cDNA was amplified in qPCR using SensiMix SYBR & Fluorescein Master Mix (Bioline) with the following primers: Me2Ex3/4 FWD ACCTTGCCTGAAGGTTGGAC, REV GCAATCAATTCTACTTTAGAGCGAAAA; CypA FWD TCGAGCTCTGAGCACTGGAG and REV CATTATGGCGTGTAAAGTCACCA. Mecp2 mRNA was normalized to Cyclophilin A housekeeping gene expression. EGFP mutants were compared with WT littermates.Frozen half brains were homogenized in Tri Reagent (Sigma) using the Ultra-Turrax T25. RNA was extracted according to the manufacturer's instructions. RNA was DNase I treated ] before adding 750 units of benzonase for 15 min at room temperature then an equal volume of 2× SDS loading buffer . Samples were boiled for 3 min before loading equal volumes on a 4–12% Run Blue SDS precast gel (Expedeon). Gels were transferred to a 0.2-μm nitrocellulose membrane (Bio-Rad) and then blocked for 1 h  before applying anti-Mecp2 1:1000 (Sigma M6818) or anti-γ-tubulin 1:3000 (Sigma T5326) overnight at 4°C, followed by IRDye 800CW donkey anti-mouse IgG 1:10 000 (LiCor) for 2 h at RT after washing. Membranes were imaged using the Odyssey Infrared Imager (LiCor) and quantified using Image Studio Lite Software (LiCor).One frozen half brain was homogenized in ice-cold NE1 buffer  and passed through a 21G needle prior to adding 750 units of benzonase then being sonicated 30 s on/off for 10 cycles . Samples were adjusted to 0.3 M NaCl, and then after centrifugation, 0.5 ml bead volume NiSO4-coated Chelating Sepharose Fast Flow beads  in lysis buffer was added to lysates for 1 h at 4°C. Beads were washed 3 times in lysis buffer before MeCP2 polypeptides were eluted with 250 mm imidazole lysis buffer in 5 fractions. Eluted fractions were pooled, adjusted to 1 mm EDTA and then dialysed [Slide-A-Lyzer Dialysis Cassette 7 K MWCO (Thermo Scientific)] overnight at 4°C in 0.1 M HEPES buffer . The polypeptides underwent a second purification step and were eluted from a HiTrap SP HP 1 ml Column  using a 0.7 M NaCl HEPES buffer. An oligonucleotide probe from the mouse Bdnf promoter region 5′ AAGCATGCAATGCCCTGGAACGGAATTCTTCTAATAAAAGATGTATCATTTTAAATGC 3′ (Biocore), plus complementary reverse strand, was methylated or unmethylated at the central CG indicated in bold italics. Five hundred nanograms of probe was radio-labelled using T4 polynucleotide kinase (NEB) and purified  according to the manufacturer's instructions. One nanogram probe and 1 μg poly deoxyadenylic-thymidylic acid competitor DNA (Sigma-Aldrich) were added to polypeptide in reaction buffer  on ice for 20 min. Samples were run at 120 V for 70 min on a 10% acrylamide tris borate EDTA gel  in chilled TBE. The gels were exposed overnight and imaged using the Typhoon FLA 9500 scanner . Percentage shift was calculated using Image J software.Recombinant MeCP2 protein expression vectors were constructed with a C-terminal histidine tag in the bacterial expression plasmid pET30b (Novagen). The R133C point mutation was introduced into the WT plasmid using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) as per the manufacturer's instructions. BL21(DE3)pLysS competent cells were transformed, and a scrape of colonies was inoculated in a starter culture overnight, then into 500 ml warm LB  and shaken at 37°C. Cultures were induced with 1 m6 HEK 293 FT cells were transfected over night with 0.5 μg of MeCP2 WT or R133C, T158M and R306C mutants tagged with EGFP, according to the manufacturer's instructions. After assessment of transfection efficiency, the medium was changed and cells were transfected with annealed unmodified or methylated oligonucleotides (100 nM) final concentration using TransIT Oligofect reagent (Mirus) for 4 h. Cells were washed with PBS and harvested by trypsinisation. In the next step, cells were cross-linked with 1% formaldehyde for 5 min at room temperature and quenched by the addition of glycine to a final concentration of 0.125 M for 5 min. At this stage, cell pellets could be flash frozen in liquid nitrogen and stored at −80°C or directly used for DNA–protein complex isolation and consecutive IP. Oligonucleotide sequence: 5′ ATGCTAATTAACCCTCACTAAAGGGAACTCGAGACATCGGAGAATTCACATCACCGGTGAATCAGTGCTACCCGCAAGTGCACTGGATCCACTGGCCGTCGTTTTACAA 3′. The transfected fragment consisted of an artificial sequence (49% GC content) flanked by T3 and M13-20 primer binding sites for unique amplification. Primer sequences are underlined. Differentially methylated CGs are highlighted in bold. Soluble chromatin preparation and chromatin immunoprecipitation assays were carried out as described previously with modifications. In short, chromatin was sonicated using a Twin Bioruptor (Diagenode) 30 s on/off for 15 cycles. Equal amounts of chromatin were used for IP with 5 μg MeCP2 M6818 antibody (Sigma) and incubated overnight. Protein–antibody complexes were bound to magnetic protein G beads (Life technologies) for 4–5 h and washed with standard IP wash buffers for 10 min at 4°C. The cross-link was reversed by addition of 0.05 volume of 4 M NaCl over night at 65°C. After proteinase K digestion, DNA was recovered by phenol–chloroform–isoamylalcohol extraction and dissolved in 200 μl H2O. Real-time PCR of ChIP DNA and corresponding input DNA was performed using T3 and M13-20 primers.Using Lipofectamine 2000 (Life technologies), 1.5 × 10m glycine. Chromatin immunoprecipitation assays were carried out as described previously with modifications. In short, chromatin was sonicated using a Twin Bioruptor (Diagenode) 30 s on/off for 15 cycles. Equal amounts of chromatin were used for IP with 40 μl GFP-Trap_A beads (Chromotek) and rotated for 1.5 h at 4°C. Protein–antibody complexes were washed with standard IP wash buffers for 4 min at 4°C. The cross-link was reversed by addition of 0.05 volume of 4 M NaCl overnight at 65°C. After proteinase K digestion, DNA was recovered by phenol–chloroform–isoamylalcohol extraction and dissolved in 200 μl H2O. Real-time PCR of ChIP DNA and corresponding input DNA was performed using specific primers. Major Satellite FWD GGCGAGAAAACTGAAAATCACG, REV AGGTCCTTCAGTGTGCATTTC; LINE1 Elements FWD TTTGGGACACAATGAAAGCA, REV CTGCCGTCTACTCCTCTTGG; IAP FWD GAGATTGGACTTTTGACTTGT, REV TGTGGCTTGCTCATAGATTAG; Bdnf FWD TTCGATTCACGCAGTTGTTC, REV CTGAGCCAGTTACGTGACCA; Cyp3a25 FWD CAGGTTTGGGGTGTTGTGAA, REV CTGCAGCTGTTGTGGGAG; Cyp3a57 FWD GTGCTGCTCTTACATGGCTG, REV GTGGGGCTACAGTCTATGCT; Cyp3a59 FWD CCTGACTGGCTGCTCACTAT, REV AGGCTGTGAACTATAGGAGCC.Frozen half brains were Dounce homogenized in 1 ml PBS and then fixed in 1% formaldehyde for 1 min at room temperature or 60 s after being on ice (stringent ChIP) before quenching with 250 mt-tests.Behavioural data were analysed using the Kolmogorov–Smirnov test with the Benjamini and Hochberg method of correction for false discovery with multiple testing. Survival data were analysed using the Mantel-Cox test. Biochemical data were analysed using two-tailed, unpaired Mice were maintained under standard conditions and in accordance with UK Home Office regulations and licenses.HMG onlineSupplementary Material is available at .Rett Syndrome Research Trust, by a Wellcome Trust Edinburgh Clinical Academic Training studentship to Kyla Brown (100670), by a Wellcome Trust Programme Grant (091580) and by a Wellcome Trust Centre Core Grant (092076). S.L. was funded by a postdoctoral EMBO long-term fellowship (ALTF 1467-2011). Funding to pay the Open Access publication charges for this article was provided by The University of Edinburgh with support from The Wellcome Trust.This work was supported by a consortium grant from the K.B. and J.S. carried out mouse behaviour, biochemical analyses and immunohistochemistry. S.L. did cellular immunoprecipitation and qPCR validation of microarrays. S.L. and K.B. did ChIP. J.C. and K.B. did EMSAs. D.S., K.B. and M.K. did mouse phenotypic scoring. J.G. designed and engineered the targeting vector. J.S. and C.M. mutated targeting vectors. J.S., J.G., K.B. and C.M. did ES cell targeting. J.S. established all mouse lines. K.B. did neuronal differentiation and microarrays. J.S. and S.L. did FACS analysis. A.K. and S.W. analysed mouse behavioural data and microarray data, respectively. A.B. and K.B. produced the manuscript.


In addition, behavioral analysis of courtship behaviors in a dyadic relationship and aggressive behaviors within a male group revealed that avt mutant males displayed decreased sexual motivation but showed normal aggression. In contrast, heterozygote V1a2 mutant males displayed decreased aggression, but normal mate-guarding and courtship behavior. Thus, impaired mate-guarding in avt and V1a2 homozygote mutants may be due to the loss of sexual motivation toward the opposite sex, and not to the loss of competitive motivation toward rival males. The different behavioral phenotypes between avt, V1a2 heterozygote, and V1a2 homozygote mutants suggest that there are redundant systems to activate V1a2 and that endogenous ligands activating the receptor may differ according to the social context.To increase individual male fitness, males of various species remain near a  mating partner and repel their rivals (mate-guarding). Mate-guarding is assumed to be mediated by two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex. The genetic/molecular mechanisms underlying how mate presence affects male competitive motivation in a triadic relationship has remained largely unknown. Here we showed that male medaka fish prominently exhibit mate-guarding behavior. The presence of a female robustly triggers male-male competition for the female in a triadic relationship . The male-male competition resulted in one male occupying a dominant position near the female while interfering with the other male's approach of the female. Paternity testing revealed that the dominant male had a significantly higher mating success rate than the other male in a triadic relationship. We next generated medaka mutants of arginine-vasotocin  mating partner and repel their rival males (mate-guarding). Mate-guarding is mediated by two different types of motivation: sexual motivation toward the opposite sex and competitive motivation toward the same sex. Here we show that the arginine-vasotocin (AVT), a non-mammalian homolog of arginine-vasopressin (AVP), system mediates the molecular mechanisms underlying how mate presence  affects male competitive motivation  in mate-guarding (a triadic relationship). We first established a novel behavioral paradigm in which medaka robustly exhibit mate-guarding behavior, that allowed us to study the genetic/molecular mechanisms underlying mate-guarding. Behavioral analysis of courtship behaviors, aggressive behaviors and mate-guarding using medaka mutants in this paradigm suggested that the AVT system is involved in the process in which mate  presence drives sexual motivation of males, which may facilitate mate guarding in the triadic relationship. Our study provides genetic evidence that the AVT/AVP system regulates mate-guarding behaviors, suggesting that the role of this system in mate-guarding is evolutionarily conserved from teleosts to mammals. Male mating strategies are considered to take two major forms: intersexual interaction  and intrasexual competition  and there is extensive literature focusing on the neural/molecular mechanisms underlying these strategies . In addiOryzias latipes), which is a commonly used model animal in molecular genetics. The medaka mating system involves socially-regulated female preference  -vasotocin . Abundant AVT peptides were detected in the wild-type (C), while no AVT peptide was detected in avt mutant (D). The x-axis shows retention time; the y-axis shows the intensity of the molecular ions.AVT peptides are present in the pituitary of the wild-type (Cab), but not in the (TIFF)Click here for additional data file.S11 FigV1a1 (V1a1+/+) and V1a1F93Y homozygotes (V1a1F93Y/F93Y) demonstrating the V1a1 T278A mutation in V1a1F93Y mutants (black arrowhead).A local sequence dataset comparing the wild-type (TIFF)Click here for additional data file.S12 FigV1a2 (V1a2+/+) and V1a2N68I homozygotes (V1a2N68I/N68I) demonstrating the V1a2 A203T mutation in V1a2N68I mutants (black arrowhead). (B) The primary structure of V1a receptor paralogs in mouse and fish. Phenylalanine 93 and arginine 68, which are identical among known forms, were changed to tyrosine and isoleucine in V1a1F93Y and V1a2N68I mutant alleles, respectively (red letters).(A) A local sequence dataset comparing the wild-type (TIFF)Click here for additional data file.S13 FigOne minute after we placed 1 fish in the tank, we calculated its movement velocity for 60 s. Mean ± SEM. Each n = 4, Dunnett’s test.(TIFF)Click here for additional data file.S14 FigavtM1R/M1R, (H) V1a2+/N68I, and (I) V1a2N68I/N68I fish. Each line indicates integrated angular velocity of five individual fish. (J) Ratio of the mean fish angular speed to that of the stripe speed. Mean ± SEM. Each n = 5, Dunnett’s test.(A) Equipment for the analysis of the OMR described previously . (B-E) R(TIFF)Click here for additional data file.S15 FigV1a1 (V1a1+/+) and V1a1 knockout (KO) homozygotes demonstrating that a 4-bp deletion generated a nonsense mutation (G26X) in V1a1 KO mutants. V1a1 gene consists of two exons. The deletion was located in the first exon and the mutated transcripts encode C-terminal deleted proteins lacked six of the seven transmembrane domains encoded by the first exon.A local sequence dataset comparing wild-type (TIFF)Click here for additional data file.S16 FigV1a2 (V1a2+/+) and V1a2 knockout (KO) homozygotes demonstrating that a 7-bp deletion generated a nonsense mutation (D24X) in V1a2 KO mutants. V1a2 gene consists of two exons. The deletion was located in the first exon and the mutated transcripts encode C-terminal deleted proteins lacked six of the seven transmembrane domains encoded by the first exon. (B) V1a1 KO mutants exhibited mate-guarding behavior, whereas V1a2 KO mutants did not. Mean ± SEM. Each n = 12, Student’s t-test: **P<0.01, ***P<0.001.(A) A local sequence dataset comparing the wild-type (TIFF)Click here for additional data file.S17 Figavt (A) and V1a2 mutants (C), but not in V1a1 mutants (B).We judged which male was closer to the female in a total 256 images  in the dominance test . Each tr(TIFF)Click here for additional data file.S18 FigV1a1F93Y homozygote mutant males exhibited normal aggressive behavior (A) and courtship behavior (B). Mean ± SEM. Each n = 8 (A), n = 11 (B), Mann-Whitney U-test.(TIFF)Click here for additional data file.S19 FigV1a2+/N68I males . Injection of AVT did not increase aggression of V1a2N68I/N68I males (E). Injection of saline did not alter aggression of the wild-type males (B), V1a2+/N68I males (D), and V1a2N68I/N68I males (F). Mean ± SEM. Each n = 6, Wilcoxon signed-rank test: *P<0.05 VS before.AVT or saline was injected to the one of three fish intraperitoneally and the number of aggressive behaviors of focal male toward other males before and after injection was counted.  Injection of AVT increased aggression of wild-type males and (TIFF)Click here for additional data file.S1 Table(TIFF)Click here for additional data file.S1 MovieThe female has the largest abdomen. The near male approaches the female and interrupts the rival  from approaching the female. This movie is played at quadruple speed.(MOV)Click here for additional data file.S2 MovieThe near male maintains its position between the female and the rival. This movie is played at quadruple speed.(MOV)Click here for additional data file.S3 MovieThe near male maintains its position between the female and the rival. This movie is played at quadruple speed.(MOV)Click here for additional data file.S4 MovieThe near male  did not interrupt the rival  from approaching the female, although its velocity is normal. This movie is played at quadruple speed.(MOV)Click here for additional data file.S5 MovieThe mutant did not interrupt the rival from approaching the female. This movie is played at quadruple speed.(MOV)Click here for additional data file.


Sepsis is characterized by capillary leakage followed by organ dysfunction. Sphingosine-1-phosphate (S1P) is a signalling lipid that regulates endothelial permeability. Here we investigated whether serum S1P concentrations are associated with sepsis severity.Primary outcome variable was the serum S1P concentration quantified by mass spectrometry. Blood was drawn at day of enrollment. S1P was correlated with inflammatory markers and Sequential Organ Failure Assessment Score (SOFA) score for sepsis severity.All three groups of patients had significantly (P < 0.001) lower serum S1P concentrations than controls . The lowest S1P concentrations was found in the septic shock group  compared to patients with severe sepsis  and sepsis . S1P levels were positively correlated with high-density lipoprotein (HDL), and red blood cell count (RBC) and negatively correlated with: procalcitonin, interleukin-6, C-reactive protein and lactate. In a multivariate linear regression model S1P, HDL and RBC were significantly associated with SOFA score (P < 0.001). S1P concentration below 158.2 µg/l was the most potent indicator for diagnosing sepsis with shock compared to all other inflammatory markers. In a multivariate logistic regression model calculated for prediction of septic shock the Odds ratio for S1P was 0.97 (CI 95% 0.96-0.99) and the best predictor of shock (P < 0.01) among all parameters tested.S1P is an indicator of sepsis severity and superior to predict septic shock than currently established markers. Further prospective studies are needed to confirm the results in a larger cohort of non-septic patients and controls.