New reassortant H5N6 highly pathogenic avian influenza viruses (AIVs) were isolated from apparently healthy domestic ducks in Southern China in 2014. Our results show that the viruses grew efficiently in eggs and replicated systemically in chickens. They were completely lethal in chicken , and the mean death time was 6 to 7 days post-inoculation. The viruses could transmit in chickens by naïve contact. BLAST analysis revealed that their HA gene was most closely related to A/wild duck/Shangdong/628/2011 (H5N1), and their NA genes were most closely related to A/swine/Guangdong/K6/2010 (H6N6). The other genes had the highest identity with A/wild duck/Fujian/1/2011(H5N1). The results of phylogenetic analysis showed that their HA genes clustered into clade 2.3.4.4 of the H5N1 viruses and all genes derived from H5 were Mix-like or H6-like viruses. Thus, the new H5N6 viruses were reassortmented of H5N1 and H6N6 virus. Therefore, the circulation of the new H5N6 AIVs may become a threat to poultry and human health. Waterfowl are a natural reservoir of avian influenza A viruses . Most su50) were calculated by the Reed–Muench method. All experiments were carried out in ABSL-3 facilities.The H5N6 HPAI viruses A/duck/Guangzhou/018/2014 (018) and A/duck/Guangzhou/021/2014 (021) were isolated from swabs of ducks in a live bird market in Guangdong, China by the Key Laboratory of Animal Disease Control and Prevention, Ministry of Agriculture of P.R. China. They were purified and propagated in 9- to 10-day-old specific-pathogen-free (SPF) embryonated hen eggs. Values of 50% egg infective doses  and transcribed into cDNA with SuperScript III reverse transcriptase (Invitrogen). PCR amplification was performed using fragment-specific primers that matched the conserved end sequence of the RNA fragments of the influenza virus. The PCR products were purified with the QIAquick PCR purification kit (QIAGEN) and sequenced using an automatic ABI Prism 3730 genetic analyzer (Applied Biosystems) according to the manufacturer’s instructions. The DNA sequences were compiled and edited using Lasergene 7.1 (DNASTAR). A phylogenetic tree of the H5N6 influenza A viruse s was generated by the distance-based neighbor-joining method using software MEGA 5 . The reliability of the tree was assessed by bootstrap analysis with 1000 replicates. Horizontal distances are proportional to genetic distance. The nucleotide sequences obtained in the present study are available from GenBank under the accession numbers (KX094400-KX094415).n = 20) were housed in isolator cages and divided into two groups with 10 per group. In each group, seven chickens were intranasally (i.n.) inoculated (106EID50 in 0.2 ml) with one of the two viruses. Three chickens were inoculated with 0.2 ml phosphate buffered saline (PBS) as negative controls. All chickens were observed for clinical symptoms for 14 days. Post-infection day 3 (DPI), three inoculated chickens in each group were euthanized to test for viral replication in the organs including the lung, kidney, brain, heart, spleen, and liver. All tissues were collected and titrated for viral infectivity in eggs. All experiments were carried out in ABSL-3 facilities in compliance with the biosafety committee of South China Agriculture University protocols. The chicken handling was performed in accordance with experimental animal administration and the ethics committee of South China Agriculture University guidelines.Six-week-old specific pathogen-free White Leghorn chickens (50)/0.1 mL. To understand the pathogenicity of the viruses in chicken, 6-week-old SPF chickens were inoculated intranasally (i.n.) with 106 EID50. All chickens were observed for 14 days after infection.In early March 2014, H5N6 AIVs—A/duck/Guangzhou/018/ 2014 (018) and A/duck/Guangzhou/021/2014 (021)—were isolated from samples of ducks as described . The virTable 1). The 018 virus replicated more highly in the lungs (6.75 log10 EID50). The mean virus titers in the heart, liver, spleen, kidney, and brain were 4.00, 5.92, 5.25, 6.58, and 5.25 log10 EID50, respectively. The 021 virus replicated in all tested organs and the highest virus titers was detected in the chicken lung .In this test, the infected chickens exhibited anorexia and depression at 1 DPI. All chickens of the 018 group died within 6 DPI. All chickens inoculated with 021 died within 7 DPI. Moreover, the viruses could transmit to chickens by naïve contact. All naïve contact chickens housed with those inoculated with 018 virus died at 4–7 DPI. Three naïve contact chickens died in the 021 group at 5–6 DPI. The viruses replicated systemically in chickens, and this could be detected in all the tested organs by 3 DPI including the heart, liver, spleen, lung, kidney, and brain  virus, and the nucleotide sequence homology was up to 97.4 and 97.6%, respectively (Table 2).All eight genes were sequenced to trace the origin and understand the genetic properties of the viruses. We performed phylogenetic analyses of these sequences and compared them with sequence data obtained from GenBank. BLASTFigure 1)  . They haigure 1) . Conservigure 1) . A previigure 1) . In our Table 2). We retrieved complete NA sequences from GenBank (including former H5N6 viruses from North America and Europe) to compare to the viruses containing the N6 gene. The H5N6 isolates belonged to the Eurasian lineage H6N6-like viruses . An 11-amino acid deletion of residues 59–69 were found in the NA genes, but not in the previous H5N6 viruses.The NA genes of the viruses had high homology with A/swine/Guangdong/K6/2010 . The genes were clustered into Mix-like viruses, which included a H5N6 virus-infected human in Sichuan in 2014 . The PB2 627K allows avian-derived viral polymerases to evade a cellular inhibitor in human cells (The PB2 and PB1 genes were most closely related to those of the A/wild duck/Fujian/1/2011(H5N1) virus , respectively, . The genes derived from Mix-like viruses included a H5N6 virus-infected human in Sichuan in 2014 . A S31N substitution of the M2 gene was associated with adamantane resistance, but this mutation was not observed in M2.The PA and M genes nucleotide sequence homology were both 98.4 and 98.8% compared with the A/wild duck/Fujian/1/2011(H5N1) virus, respectively, (Table 2). The NP and NS genes were derived from the Mix-like viruses that included a H5N6-infected human in Sichuan in 2014 . The 678 amino acid residue of PB1 was S, the 615 amino acid residue of PA was K, and the 319 amino acid residue of NP was N. This indicated no genetic markers associated with increased virulence in mammals (The NP and NS genes were highly homologous with the viruses A/hill myna/Heilongjiang/0704/2012 (H5N1) and A/wild duck/Fujian/1/2011(H5N1), respectively,  and 021 (H5N6)] were isolated from apparently healthy domestic ducks in early March 2014. They were completely lethal in chickens , and the mean death time (MDT) was 0–7 DPI. The viruses could also cause horizontal transmission in chickens by naïve contact. All the naïve contact chickens in these two groups show the same clinical signs as infected chickens, and the MDT was 6 or 7 DPI. Moreover, the two viruses could replicate systemically in chickens. Virus was found in all the tested organs at high titer levels—especially the lung. The short incubation period and rapid progression of the H5N6 viruses were consistent with HPAI. Thus, our findings showed that the H5N6 viruses from waterfowl were highly pathogenic to chickens and could transmit among them.Their HA genes were Mix-like and were assigned to clade 2.3.4.4. Their NA genes were derived from H6N6-like Eurasian lineage viruses; the internal genes included M, NS, NP, PA, PB1, and PB2 derived from Mix-like viruses. In addition, the H5N6 virus-infected human in Sichuan in 2014 also belongs to the Mix-like viruses. Such Mix-like gene pools are characterized by H5N1 and H5N6 subtypes of AIVs isolated from Vietnam and Laos. In addition, a small amount of H5N1 and H5N2 viruses were isolated in China. The H5N1 gene pool has widened and emerged since 2003.Figure 3). Moreover, the data suggested that the HA genes and the internal genes of H5N1 viruses from Vietnam are highly related to those from the H5N6 viruses. Therefore, our results suggested that the H5N6 viruses might circulate among Southern China, Vietnam and Laos.In the future, the community must study whether this Mix-like gene pool will become more diverse and even branch into a new subtype/genotype. Moreover, the H5N6 avian influenza broke out in Laos in late March 2014. The highly homologous nucleotide in our study suggested that our isolates might have an identical origin with the A/duck/Laos/LPQ001/2014(H5N6) virus from Laos. The NA genes of our isolates and the LPQ001 virus were highly homologous with the virus A/swine/Guangdong/K6/2010(H6N6). The other genes of our viruses and the LPQ001 virus were highly homologous with the H5N1 viruses from Fujian, respectively, (Reassortant among different influenza viruses is considered the main mechanism for the emergence of new viruses. The co-circulation of H5 and H6 influenza viruses in domestic ducks contributed to the antigenic and genetic diversity of influenza viruses in Southern China. Zhejiang, Hubei, and Guangdong in particular had an outbreak of H5N6 avian influenza in farms in 2014 followed by Hunan and Jiangsu in 2015 . In addiPJ and JC designed this study, performed the experiments and participated in the data collection and analysis. YS, HS, ZZ, QY, SW, NQ, NW, and GO assisted with animal experiment. PJ and JC drafted the manuscript. ML participated in writing the discussion.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Modified on a cheap glass carbon electrode, these MnFe2O4/Au hybrid nanospheres show favorable sensitivity (0.315 μA/ppb) and limit of detection (LOD) (3.37 ppb) toward As(III) under the optimized conditions in 0.1 M NaAc-HAc (pH 5.0) by depositing for 150 s at the deposition potential of −0.9 V. No obvious interference from Cd(II) and Hg(II) was recognized during the detection of As(III). Additionally, the developed electrode displayed good reproducibility, stability, and repeatability, and offered potential practical applicability for electrochemical detection of As(III) in real water samples. The present work provides a potential method for the design of new and cheap sensors in the application of electrochemical determination toward trace As(III) and other toxic metal ions.Au nanoparticles decorated mesoporous MnFe Such po 10 ppb) ,4. Thus,required ,6. Howevrequired ,8,9. Therequired ,10. So frequired ,12. Howerequired ,13. It wrequired ,15,16.3O4, as a low-cost, eco-friendly, and easily-prepared material with ideal catalytic and absorption activity, has attracted great attention for its high sensitivity and rapid response toward arsenic via the electrochemical method [3O4 microspheres composited with room-temperature ionic liquids (RTILs) for the determination of As(III), which reached an exciting electrochemical performance  for the detection of As(III) through square wave anodic stripping voltammetry (SWASV) compared with the noble-metal nanoparticle modified electrodes [3O4 nanocrystals were also reported that could be exploited in order to remove arsenic from water due to their strong adsorption capacity [2 plays a role as an oxidant in the As(III) uptake by FMBO and MNFHO, which could perform much better than pure Fe oxides for the removal of As(III). [et al. reported the MnFe2O4 combined with uniform mesoporous structured and small constituent nanocrystals can provide a synergistic effect for the enhanced adsorption performance toward As(III) [2O4 nanocrystal clusters exhibited favorable sensing behavior toward As(III) with the sensitivity of 0.295 μA/ppb and LOD of 1.95 ppb by SWASV [Recently, metal-oxide nanomaterials have been creatively introduced into voltammetric responses for inorganic arsenic due to their excellent catalytic activity and adsorption ability ,18,19. Fl method . Gao repcapacity . To enhacapacity . NoteworAs(III). ,23. Xu e As(III) . After bby SWASV . Thus, i2O4 nanocrystal clusters (MnFe2O4/Au hybrid nanospheres) for electrochemical sensing of As(III) by square wave anodic stripping voltammetry (SWASV) [2O4/Au hybrid nanospheres were characterized and studied. The optimization of detection conditions—including considerations regarding supporting electrolytes, pH values, deposition potentials, and deposition times—were investigated. Moreover, electrochemical interference behavior, reproducibility, stability, repeatability, and real sample analysis of the MnFe2O4/Au hybrid nanospheres modified GCE were also studied in detail under the optimum experimental conditions.In this work, considering the high electrochemical response of Mn doped iron oxide and the excellent electrocatalytical ability of Au nanoparticles towards As(III), we have explored Au nanoparticle decorated mesoporous MnFe (SWASV) ,27. A gl3PO4, KH2PO4, K2HPO4, and NaOH. NH4Cl-NH3·H2O (0.1 M) solutions were prepared by mixing stock solutions of 0.1 MNH4Cl and NH3·H2O in different proportions. All stock and working solutions were prepared in purified water (18.2 MΩ cm), which was obtained through a GWA-UN to Pure & Ultrapure water purification system .All chemicals were analytical grade, and were commercially available from Shanghai Chemical Reagent Co. Ltd without further purification. Acetate buffer solutions of 0.1 M at different pH values were prepared by mixing stock solutions of 0.1 M NaAc, HAc, and NaOH. Phosphate buffer solutions (PBS) of 0.1 M were prepared by mixing stock solutions of 0.1 M HThe Electrochemical measurements were performed on a CHI 660E computer-controlled potentiostat . A conventional three-electrode configuration was employed, consisting of a bare or modified glass carbon electrode (GCE) as a working electrode, an Ag/AgCl/saturated KCl electrode as a reference electrode, and a platinum wire as a counter electrode. Scanning electron microscopy (SEM) observation and energy-dispersive X-ray spectrum (EDS) analyses were carried out on a Quanta 200 FEG scanning electron microscope . The High Resolution Transmission Electron Microscopy (HRTEM) and Transmission Electron Microscopy (TEM) images analyses were carried out on JEM-2010 and JEM-1400 microscopes , respectively.2O4 nanocrystal clusters ) were prepared according to a previous report [2O4 NCs (20 mg) were added to a solution containing ethanol (30 mL) and water (2 mL), followed by the addition of ammonium hydroxide  and APTMS (200 μL). Then, the resulting solution was sonicated continuously for about 8 h. After four-step separation by means of an external magnetic field, the resulting APTMS-functionalized MnFe2O4 NCs was dissolved in water (10 mL). After being selected with a magnet and rinsed with water four times, the resulting APTMS-functionalized MnFe2O4 NCs were dissolved in water (10 mL) for further use. Firstly, the mesoporous MnFes report . Then ths report : MnFe2O42O4 NCs (1 mL) was mixed with 20 mL solution of Au nanoparticles via shaking for 24 h. The solution of Au nanoparticles was synthesized according to previously described methods [2O4/Au hybrid nanospheres was collected through an external magnet. An illustration of the preparation procedure of mesoporous MnFe2O4/Au hybrid nanospheres can be followed in Subsequently, the solution of APTMS-functionalized MnFe methods . The res3 solution, absolute ethanol, and water for 1 min, respectively. Subsequently, the mesoporous MnFe2O4/Au hybrid nanospheres film on the surface of the GCE was produced as follows: Mesoporous MnFe2O4/Au hybrid nanospheres (2 mg) were dispersed into purified water (2 mL) to obtain a uniform dispersion by 3 min of sonication. A drop of the above MnFe2O4/Au hybrid nanospheres solution was pipetted onto the fresh surface of the GCE and dried in air. After evaporation, a thin mesoporous MnFe2O4/Au hybrid nanospheres film was formed on the surface of the GCE.Firstly, the GCE was polished with 0.3 μm and 0.05 μm alumina power slurries sequentially to form a mirror-shiny surface. Next, it was sonicated with 1:1 HNO2O4/Au hybrid nanospheres modified GCE was observed through square wave anodic stripping voltammetry (SWASV) under the optimized conditions. Under the potential of −0.9 V for 150 s, As(0) was deposited by the reduction of As(III) in 0.1 M HAc-NaAc (pH 5.0). Subsequently, the electrodeposited As(0) was anodic stripped to As(III) which was performed in the potential range of −0.4 to 0.4 V under the following conditions: frequency, 25 Hz; amplitude, 25 mV; increment potential, 4 mV; vs. Ag/AgCl. Thereafter, desorption potential of −0.9 V for 150 s was carried out to remove the residual As(0) under stirring conditions. The same experimental conditions were applied in the interference, reproducibility, stability, repeatability, and real sample analysis studies. Cyclic voltammograms (CV) and electrochemical impedance spectra (EIS) were performed in the mixing solution containing 5 mM Fe(CN)63−/4− and 0.1M KCl with the scanning rate of 100 mV∙s−1.The electrochemical behavior for the MnFe2O4 sample is made up of monodisperse, uniform, and smooth-faced microspheres with diameters of about 350 nm. The HRTEM image indicates that the individual microsphere contains lots of loose clusters, which are composed of primary nanocrystals with the size of about 8–12 nm . 8–12 nm b. This m 8–12 nm b, also s2O4 NCs, and MnFe2O4/Au hybrid nanospheres modified GCE were electrochemically characterized in a solution of 5 mM Fe(CN)63−/4− with 0.1 M KCl  decreases from 190 mV to 120 mV, indicating the electron transfer of MnFe2O4 NCs were improved observably by the hybrid of the Au nanoparticles on the surface of MnFe2O4 NCs.The typical cyclic voltammetry (CV) of bare GCE, Au, MnFe.1 M KCl a. Both ttransfer . However2O4 NCs, and MnFe2O4/Au hybrid nanospheres modified GCE are further analyzed by electrochemical impedance spectroscopy (EIS). The diameter of the semicircle equals the electron transfer resistance (Ret), and the Ret controls the electron transfer kinetics of the redox probe at the surface of the electrode. et for this modified electrode. However, et of the MnFe2O4 NCs modified GCE was much higher than the bare or Au nanoparticles modified GCE, indicating that the MnFe2O4 NCs layer blocked the electron transfer of the electrochemical probe. Due to the poor conductivity of metal oxide, the modification of MnFe2O4 NCs could further pose a barrier and hinder the access of the redox probe to the electrode surface, thus resulting in a large Ret of the MnFe2O4 NCs modified GCE. Interestingly, the EIS of the MnFe2O4/Au hybrid nanospheres modified GCE shows a much smaller semicircle compared with the MnFe2O4 NCs modified GCE, indicating that the decorating of Au nanoparticles on the surface of MnFe2O4 NCs could improve the electron transfer of MnFe2O4 NCs on the surface of the GCE, which is consistent with the above CV results.The interface properties of the bare GCE, Au, MnFe2O4 NCs, Au nanoparticles, and MnFe2O4/Au hybrid nanospheres modified GCE. When the accumulation process was performed for 150 s at −0.9 V in 50 ppb As(III) solution containing 0.1 M acetate buffer (pH 5.0), it shows no peak appears in the curve of the MnFe2O4 NCs modified GCE, and a weak peak at −0.9 V can be observed for the Au nanoparticles modified GCE. For the MnFe2O4/Au hybrid nanospheres modified GCE, the peak current increases much higher than the single MnFe2O4 NCs or Au nanoparticles modified GCE. It shows that the hybrid that consists of Au nanoparticles on the surface of MnFe2O4 NCs could provide much more remarkable electrochemical performances towards As(III) detection. It might be attributed to the catalytic effect and high electron transfer of Au nanoparticles would provide much more active sites for the electrochemical reactions in the detection process of As(III). Thus, based on the unique mesoporous structure of the MnFe2O4 NCs responses to As(III) [2O4 NCs indeed provides a synergistic effect for the detection of As(III) through SWASV. As(III) , the hyb4Cl-NH4OH  decreased under higher pH values [2O4 NCs. Thus, pH 5.0 was chosen for the preconcentration solution in this experiment. Different deposition potentials in the range of −1.2 to −0.4 V were also experimented with via SWASV by standard additions of 50 ppb As(III) in 0.1 M NaAc-HAc (pH 5.0), as shown in vs. Ag/AgCl) can obviously improve the peak current of reduction of As(III). Generally, the peak current would reach a plateau, settling to a constant value which should be the optimum deposition potential. However, when the potentials beyond −0.9 V were applied, a decrease response of the peak current were observed, as shown in The optimization of experimental conditions  was taken into account using SWASV. The effect of supporting electrolytes at pH 5.0 was tested and NaAc-HAc was finally chosen as the best electrolyte in comparison with PBS and NHCl-NH4OH a. The efH values . In a hi2O4/Au hybrid nanospheres modified GCE using SWASV. 3O4-RTIL modified SPCE [3O4-RTIL composite could provide a specific interface for arsenic to accumulate and exchange electrons, thus future studies focusing on the MnFe2O4-Au-RTIL composite may obtain exciting sensitivity and LOD for the electrochemical sensing of As(III).Under the optimal experimental conditions, As(III) was determined on the MnFeied SPCE , the sen2O4/Au hybrid nanospheres modified GCE in 0.1 M HAc-NaAc (pH 5.0) containing 50 ppb As(III) in the presence of Cd(II), Hg(II), Pb(II), and Cu(II) and over a concentration range of 0 to 500 ppb, respectively. 2O4/Au hybrid nanospheres modified GCE when sensing the two target metal ions simultaneously. As to the interference of Cu(II), 2O4/Au hybrid nanospheres modified GCE when the concentration of Cu(II) is beyond a certain value when the Cu(II) and As(III) coexist within a solution. On the other hand, Commonly, the other metal ions can coprecipitate and strip off under the experimental conditions for determination of As(III), thus sensitive detection of As(III) in the real sample without interference is a challenging task. In order to investigate the interference of other heavy metal ions to the electrochemical determination of As(III), a series of interference measurements were studied. 2O4/Au hybrid nanospheres modified GCEs were prepared following the same procedure and were applied to detect 50 ppb As(III) in 0.1 M HAc-NaAc (pH 5.0) under the optimized conditions. The relative standard deviation (RSD) derived from the peak currents from six tests was 3.9%, indicating that the developed method has good reproducibility. Additionally, the stability of the modified electrodes were investigated by storing six prepared electrode at 4 °C for ten days, and then testing the SWASV response toward 50 ppb As(III) under the optimized conditions. The result showed that the stripping peak current only decreased by 7.8%, 7.3%, 3.7%, 4.6%, 7.0%, and 4.8% of the first value, thereby showing a long-term stability of the fabricated electrode. Moreover, the repeatability of the MnFe2O4/Au hybrid nanospheres modified GCE were also evaluated by conducting repetitive experiments for 20 times to detect 50 ppb As(III) on the modified electrode under the optimized conditions. 2O4/Au hybrid nanospheres modified GCE make them attractive for the preparation of a electrochemical sensor toward As(III).To evaluate the reproducibility of the modified electrode, six MnFe2O4/Au hybrid nanospheres modified GCE, real water sample analyses were taken from laboratory tap water [In order to evaluate the practical application of the MnFeap water ,42. The 2O4 nanocrystal clusters (MnFe2O4/Au hybrid nanospheres) with high electrochemical performance were used for the electrochemical determination of As(III) by square wave anodic stripping voltammetry (SWASV). The as-prepared product was characterized by SEM, HRTEM, TEM, and EDS. The results suggest that 13 nm Au nanoparticles were indeed supported on the surface of mesoporous MnFe2O4 microspheres with the diameter of about 350 nm. Electrochemical behavior of the MnFe2O4/Au hybrid nanospheres modified GCE toward As(III) showed favorable sensitivity (0.315 μA/ppb) and LOD (3.37 ppb) for As(III) under the optimized conditions in 0.1 M NaAc-HAc (pH 5.0) by depositing for 150 s at the deposition potential of −0.9 V. No obvious interference from Cd(II) and Hg(II) was recognized during the detection of As(III). In addition, the excellent reproducibility, stability, and repeatability of MnFe2O4/Au hybrid nanospheres made it a promising electrode material for the electrochemical determination of As(III). Furthermore, the MnFe2O4/Au hybrid nanospheres modified GCE offered potential practical applicability in the electrochemical detection of As(III) in real water samples. Ultimately, the present work may provide a potential method for the design of new and cheap sensors in the application of electrochemical detection toward trace As(III) and other toxic metal ions.In summary, Au nanoparticles decorated mesoporous MnFe


Arabidopsis thaliana, shoot meristem stem cells are maintained by the homeobox transcription factor gene WUS (WUSCHEL) that is expressed in cells of the organizing center underneath the stem cells. In order to identify factors that operate together with WUS in stem cell maintenance, we performed an EMS mutant screen for modifiers of the hypomorphic wus-6 allele. We isolated the oberon3-2 (obe3-2) mutant that enhances stem cell defects in wus-6, but does not affect the putative null allele wus-1. The OBE3 gene encodes a PHD (Plant Homeo Domain) protein that is thought to function in chromatin regulation. Single mutants of OBE3 or its closest homolog OBE4 do not display any defects, whereas the obe3-2 obe4-2 double mutant displays broad growth defects and developmental arrest of seedlings. Transcript levels of WUS and its target gene in the stem cells, CLAVATA3, are reduced in obe3-2. On the other hand, OBE3 and OBE4 transcripts are both indirectly upregulated by ectopic WUS expression. Our results suggest a positive feedback regulation between WUS and OBE3 that contributes to shoot meristem homeostasis.The stem cells in the shoot apical meristem (SAM) are the origin of all above ground tissues in plants. In  Arabidopsis thaliana. The homeodomain transcription factor WUS is expressed in the organizing center (OC) underneath the stem cells [WUS transcription via CLV1/CLV2-CRN receptor-like kinases to delimit the size of the OC [Postembryonic growth and iterative organ formation of higher plants rely on the activity of pluripotent stem cells in organogenic centers, the meristems. The shoot meristem that will give rise to the above ground organs has been extensively studied in the model plant em cells  where item cells  and, aftem cells , 4, reprem cells . CLV3 inf the OC –8. This f the OC . The WUSf the OC , 7. In cf the OC –11. In af the OC –14. HoweAlthough in the recent years, many studies identified further components affecting WUS/CLV3 homeostasis –23, how wus-6 allele [wus enhancer 9 (wen9) mutant that enhances stem cell defects in wus-6. We show by positional cloning that wen9 is an allele of the OBE3 gene, and characterize its function together with its closest homologue OBE4 in the shoot meristem.In order to find hitherto undiscovered factors involved in the WUS-mediated stem cell regulation, we used a sensitized mutant screen for genetic modifiers of the hypomorphic 6 allele , 24. Herwus-1 causes premature termination of stem cells in the primary shoot meristem during embryogenesis, resulting in a flat apex of partially differentiated cells at the seedling stage [wus-6 allele causes reduced WUS expression levels, and the primary seedling shoot meristem and floral meristem prematurely terminate indistinguishably to wus-1 , was mapped to a 97 kb region between position 5001124 and 5098789 on chromosome 1, and a nonsense mutation was identified in the predicted first exon of locus AT1G14740. The encoded protein OBERON3 (OBE3) [wen9 mutation introduces a stop codon after the PHD domain at amino acid position 518. OBE3 transcript levels are reduced in wen9 to about half of the wild-type level  , also na3 (OBE3) , is 733 pe level . Transfofragment  suppress6 plants ; S4 Fig r extent ; S5 Fig.WUS and OBE3, we analyzed double mutants between different wus alleles and the obe3-2 mutant. Development of the homozygous obe3-2 single mutant is indistinguishable from wild type  pattern. In cases where flowers are made, obe3-2 does not further enhance the already early termination of wus-1 and wus-6 floral meristems, but causes premature termination of wus-7 floral meristems  and floral meristems (wus-7).In summary, residual obe3-2 single mutant does not display any developmental defect, we asked whether related genes might mask its function. To this end, we isolated an insertion mutant in the closest OBE3 homolog OBE4, also named TITANIA2 (TTA2) [obe4-2 (SAIL_827_F11) mutation disrupts exon1, suggesting that it is a severe loss of function allele  . The oben allele . OBE4 trpe level . obe4-2 ild type .obe3-2/+ obe4-2/+ mother plant, we found 2.8% (n = 143) very small seedlings with partially fused cotyledons and without any true leaves, did not develop further, and were genotyped as homozygous double mutants by PCR  formed true leaves, but display severe growth retardation  and that all double mutant plants (n = 6) were mosaics carrying both, wild-type-like complete flowers (40/170) and wus-1-like incomplete flowers  compared to the Ler wild type, whereas ARR7 mRNA levels appear increased  inflorescence and floral meristems unlike in the wild type  whereas seedlings with weak shoot meristem defects are increased . By contrast, defective wus-1 flower development is not altered by p35S:cOBE3  with an enhanced phenotype. The wen9 mutation was mapped with SSLP and dCAPS markers to a 97 kb region in chromosome 1 (between SNP CER465614 and CER424346). Sequencing of all 23 candidate loci in this region detected a mutation only in the OBE3 gene. The identified G1554>A “stop” mutation is in exon1 of the predicted reading frame of locus AT1G14740. The primers wen9-F (5’- CAGAGATGTTTGGATTCGTTAAGGATGTTTTTGTGTGTTGCGCTAAGAATCG-3’) and wen9-R (5’-GAAATTGTGATAAGAGAAGG-3’) were used for genotyping PCRs (Ta 55°C). After TaqI restriction cleavage, the wild type displays a 300bp band, and wen9 displays a 250bp and a 50bp band. Genotyping primers of other mutants including T-DNA lines used in this study are listed in The OBE3 was amplified by PCR from Ler and cloned as pOBE3:gOBE3 in a pGPTV-HPT-based vector. The RALF11-03K20 cDNA clone from RIKEN BRC was used to amplify the full length cDNA by PCR for construct preparation. The cDNA fragments starting from ATG to the end of gene, with or without the stop codon, were cloned as: p35S:cOBE3, and p35S:cOBE3-GR respectively, in a pGreenII-based vector. Arabidopsis plants were transformed by floral dip, and T1 seeds were selected on plates with the respective antibiotics.The genomic fragment including the intergenic region of Arabidopsis seeds were surface-sterilized with 1% hypochlorite for 10 minutes and washed two times with 70% ethanol. Sterilized seeds were sown on 1/2 MS plates, stratified for 3 days in the dark at 4°C and then grown in a Percival growth cabinet with constant illumination for 7 or 10 days. For all qRT-PCR experiments, 3 biological replicates with two technical replicates each were done.For experiments without further treatment, seedlings were collected from the plates and frozen in liquid nitrogen immediately. For the induction experiments, dexamethasone (5 μM), with or without cycloheximide (50 μM) were applied by spraying the plates, and flooding the seedlings for 15 minutes. After removal of the liquid, seedlings were returned to the Percival growth cabinet for 4 hours before sample collection.® Mini kit (QIAGEN), followed by RQ1 RNase-Free DNase (Promega) treatment and reverse-transcribed with SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). Quantitative PCR was performed with the LightCycler® 480 system (Roche) coupled with SYBR Green I Master (Roche). For each qPCR reaction, 25 ng of cDNA was used. Transcript level analysis was carried out according to a published protocol [t-tests were performed on Cp’ values. The Cp’ values were calculated after normalizing the Cp values with three independent reference genes, which passed the geNorm v3.5 [Total RNA was extracted from whole seedlings using the RNeasyprotocol . For staorm v3.5  test. FoS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.


Previously, we demonstrated that stigma towards persons with migraine was comparable in magnitude to stigma towards individuals with epilepsy or panic disorder.We sought to measure stigma towards individuals with differing migraine phenotypes versus comparator conditions.Subjects were recruited on-line (MTurk), consented, and randomized to assess one of six fictional vignettes:1) a woman with migraine four days/month with zero lost workdays/year (W0)2) a woman with migraine four days/month with two lost workdays/year (W2)3) a man with migraine four days/month with two lost workdays/year (M2)4) a woman with seizures four days/month with two lost workdays/year (E2)5) a woman caring for her invalid husband four days/month with two lost workdays/year (H2)6) a woman with migraine twenty days/month with ten lost workdays/year (W10)Subjects used sliders from 0 to 100 to answer five questions measuring social distance attitudes (SDA) towards the individual described in the vignette; maximum stigmatizing attitude would be a total SDA score of 500. [Study approved by UBC Office of Research Services.]3,617 total US subjects ≥19 years old completed the survey .W2/M2/E2 scores and W0/E2 scores did not differ significantly. Relative to W2/M2, W0 was significantly lower and H2 was significantly higher. W10 was significantly higher than H2.Among MTurk subjects, stigma towards persons with migraine increased with their absenteeism, but did not vary by gender.No conflict of interest.


Saccharomyces cerevisiae demonstrate a two-step DSB resection, wherein CtIP and Mre11 nucleases carry out short-range DSB resection followed by long-range DSB resection done by Dna2 and Exo1 nucleases. Recent studies indicate that CtIP contributes to DSB resection through its non-catalytic role but not as a nuclease. However, it remains elusive how CtIP contributes to DSB resection. To explore the non-catalytic role, we examined the dynamics of Dna2 by developing an immuno-cytochemical method to detect ionizing-radiation (IR)-induced Dna2-subnuclear-focus formation at DSB sites in chicken DT40 and human cell lines. Ionizing-radiation induced Dna2 foci only in wild-type cells, but not in Dna2 depleted cells, with the number of foci reaching its maximum at 30 minutes and being hardly detectable at 120 minutes after IR. Induced foci were detectable in cells in the G2 phase but not in the G1 phase. These observations suggest that Dna2 foci represent the recruitment of Dna2 to DSB sites for DSB resection. Importantly, the depletion of CtIP inhibited the recruitment of Dna2 to DSB sites in both human cells and chicken DT40 cells. Likewise, a defect in breast cancer 1 (BRCA1), which physically interacts with CtIP and contributes to DSB resection, also inhibited the recruitment of Dna2. Moreover, CtIP physically associates with Dna2, and the association is enhanced by IR. We conclude that BRCA1 and CtIP contribute to DSB resection by recruiting Dna2 to damage sites, thus ensuring the robust DSB resection necessary for efficient homologous recombination.Homologous recombination plays a key role in the repair of double-strand breaks (DSBs), and thereby significantly contributes to cellular tolerance to radiotherapy and some chemotherapy. DSB repair by homologous recombination is initiated by 5’ to 3’ strand resection (DSB resection), with nucleases generating the 3’ single-strand DNA (3’ssDNA) at DSB sites. Genetic studies of  DSBs are generated during physiological replication and can be induced by chemotherapeutic agents such as camptothecin, cisplatin and poly[ADP ribose]polymerase (PARP) inhibitors, as well as by IR. DSBs are repaired by the two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR, but not NHEJ, repairs DSBs induced by chemotherapeutic agents as well as those that occur during physiological replication , 2. HR cSaccharomyces cerevisiae, DSB resection is carried out by four nucleases: Mre11-Rad50-Xrs2 (the MRX complex), Sae2, Dna2 and Exo1 [HR is carried out in a series of steps, beginning with DSB resection, specifically the 5’ to 3’ strand resection of DSBs . The resand Exo1 . The MRXand Exo1 , 11. MRXand Exo1 . Dna2 anBRCA1-/- cells is restored to near normal by the additional inactivation of 53BP1, a protein involved in NHEJ [Recent studies have suggested that yeast Sae2 and mammalian CtIP contribute to DSB resection in a different manner. Unlike Sae2, CtIP plays the central role in DSB resection, as inactivation of CtIP as well as Mre11 is mortal to cells , 13, and in NHEJ , 20.BRCA1-/- cells also displayed a defect in Dna2-focus formation whereas BRCA1-/-/53BP1-/- cells displayed nearly normal Dna2 foci. We propose that 53BP1 and BRCA1 may control the CtIP—dependent recruitment of Dna2 to DNA damage sites for subsequent DSB resection.We found that the inactivation of nuclease activity associated with Dna2 completely inhibited DSB resection, as did the depletion of CtIP in both human cells and chicken DT40 cells , 21. Dep2 in RMPI-1640 medium  with glutamine  supplemented with 1% chicken serum , 10% heated-inactivated fetal bovine serum (FBS) , 50 μM mercaptoethanol (Invitrogen), 50 U/ml penicillin and 50 μg/ml streptomycin . Doxycycline was added at a final concentration of 100 ng/ml to inactivate the expression of the tetracyclin repressible promoter.The DT40 cell line is derived from chicken B lymphoma  and was 2 in DMEM media  supplemented with 10% FBS (Gemini Bio-Products) for U2OS and MCF7 cell lines or in RPMI-1640  with 10% FBS (Gemini Bio-Products) for HCC1937 cell lines.Human cell lines  were maintained at 37°C with 5% CODNA2 knockout construct, genomic DNA was amplified with the following primers: F1 and R1 (2.9 kb) for the left arm and F2 and R2 (2.7 kb) for the right arm. Each product cloned was combined on pBLUESCRIPT-SK (pBS-SK) plasmid, after which the histidinol dehydrogenase (hisD) gene was introduced with BamHI. To generate the D245A  knockin construct, the mutation was introduced by polymerase chain reaction (PCR) using two sets of primers: F1, F3, R3, and R1. The knockout and knockin constructs were digested with XhoI for linearization. Linearized plasmid (30 μg) was transfected into 107 cells. The genomic DNA was digested with StuI and BamHI for Southern blotting. 0.6 kb DNA fragment amplified with F4 and R4 from genomic DNA was used as probes for Southern blotting. The expression of DNA2-/D245A transcript was confirmed by RT-PCR using F5 and R5. Each amplicon, 430 bp for D245A fragment, was digested using BstUI. To construct a plasmid for the inducible expression of DNA2, cDNA2 was cloned downstream from the tetracycline promoter with SalI and BamHI. Transfection for chicken DT40 cells was performed as previously described [All primers used for plasmid construction are shown in escribed .CtIP knockout construct, genomic DNA was amplified with F6 and R6 for the left arm, which was cloned in pBS-SK with NotI and BamHI. To generate the N183A,R187A knockin construct, the mutation was introduced by PCR using two sets of primers: F7, F8, R7, and R8 and the product was cloned into pBS-SK with BamHI and XhoI. The construct was digested with XhoI for linearization. The expression of CtIPN183A,R187A/-/- transcript was amplified by RT-PCR using F9 and R9 and digested with BfaI.To generate a Cells were counted and cell-cycle analysis was carried out using a flow cytometer . Immediately before being counted, cells were mixed with reference beads  and propidium iodide. Cells were treated with BrdU  for 10 minutes and then fixed with 70% EtOH for the cell-cycle analysis. Cells were then incubated with FITC-conjugated anti-BrdU  and propidium iodide. Fluorescence data were displayed as dot plots using Cell Quest and Diva software. HeLa cells were synchronized using a double thymidine block as described previously . In briesiRNA oligos were purchased from QIAGEN (CtIP) and Dharmacom . Transfection was done using Lipofectamine 2000 (Life Technologies) according to the attached protocol. After transfection, cells were cultured in fresh media for 48 hours prior to being harvested for Western blotting and immunostaining assays.in vivo, cells (1 x 107 cells for U2OS and ATLD2) exposed to 6 Gy γ-ray irradiation were incubated for 1 hour. Lysate was prepared from cells pretreated with paraformaldehyde  for 10 minutes before collection. The lysis buffer contained HEPES-KOH (ph7.5), 300mM KCl, 0.1% Triton X-100, and protease cocktail . CtIP and Dna2 were detected using anti-rabbit IgG conjugated with HRP .Human DNA2 and CtIP were detected using anti-CtIP  and anti-DNA2 . To detect protein interaction Cells were treated with 0.1 μg/ml colcemid  for 3 hours. Cells were suspended in 75mM potassium chloride for 15 minutes, washed with Carnoy’s solution (a 3:1 mixture of methanol and acetic acid), dropped on slides, and stained with a 5% Giemsa solution for 10 minutes.4P2O7, 1 mM Glycerol-ß-phosphate, 5 mM PMSF. Lysate was sonicated on ice until homogenous and insoluble material removed by centrifugation  for one hour. The supernatant was bound to 2 ml of anti-Flag M2 agarose resin slurry (Sigma) at 4°C for one hour followed by washing on a column with 20 ml buffer A, 10 ml 0.5 M LiCl, and 20 ml buffer A. The protein was eluted with 10 ml buffer A containing Flag peptide (Sigma) at a concentration of 100 μg/ml. Eluted protein was loaded onto a 1 ml Hitrap SP column (GE), washed with 20 ml buffer A and eluted with buffer A containing 0.6 M NaCl. The peak fractions were dialyzed twice against fresh 300 ml buffer A for one hour and flash frozen in liquid nitrogen. Dna2 protein was expressed in Sf21 insect cells using the baculovirus expression system. Dna2 expressing cells were lysed by homogenization and sonicated three times for 20 seconds in A buffer containing 0.5% tween-20 and 1mM PMSF (phenylmethylsulfonyl fluoride). The lysate was centrifuged for 1 hour at 35,000 rpm at 4°C. The supernatant was incubated with ~1ml M2 anti-Flag antibody-conjugated agarose resin (Sigma) with rotation at 4°C for 1 hour. After incubation the lysate with resin was centrifuged for 3 min at 1000g. After removing the supernatant, the remaining resin was washed with 20 ml of A buffer twice and was eluted with 5 ml of A buffer containing 0.8mg/ml 3X Flag peptide (Sigma). The peptide was incubated with the resin for 20 min before elution. The Flag eluent was then loaded onto 1 ml HiTrap SP column (G.E.) and washed with buffer A then eluted with buffer A containing 500 mM NaCl. The eluted protein fractions were dialyzed in A buffer and the dialyzed fractions were aliquoted, frozen in liquid nitrogen, and stored at -80°C. Purified recombinant CtIP and Dna2 were incubated on ice for 1 hour in a 100 μl reaction containing buffer A. After incubation, CtIP was immunoprecipitated using 5 μg of anti-CtIP antibody (Bethyl) and 10 μl of Protein A resin (Sigma). The beads was washed 3 times with buffer A and separated on a 6% SDS-PAGE. The gel was transferred onto a PVDF membrane, which was blotted against anti-Dna2 (Pierce: PA5-23691), and further visualized with horseradish peroxidase-conjugated anti-rabbit secondary antibody by chemiluminescence (Pierce).Human CtIP purification from insect cells: Cells were lysed in buffer A  supplemented with 0.5% Triton, 2.5 mM NaAfter harvest, cells were fixed by 4% paraformaldehyde for 10 minutes, followed by permeabilization using 0.5% NP-40 in PBS for 10 minutes. Fixed cells were treated with anti-Rad51  and FITC-conjugated anti-rabbit . Rad51 foci were counted in a minimum of 100 cells were visualized at the indicated times. Dna2  and γH2AX  foci were visualized by anti-rabbit Alexa Fluor 594  and anti-mouse Alexa Fluor 488 , respectively, then analyzed using a BZ-9000 fluorescence microscope . The number of Dna2 and γH2AX foci was automatically counted using BZ-Analyzer-II software (Keyence). Signals over 0.1 square μm on each nucleus were extracted and all images were normalized with the same value to cut off background signalsDNA2 gene is located on chromosome VII  and encodes a 992 amino-acid protein. To explore the function of Dna2, we generated DNA2-gene-disruption constructs to remove seven exons encoding 282 to 630 amino acids  into the DNA2+/- cells (DNA2+/-/tet-DNA2). We then deleted the second DNA2 allele to generate DNA2-/-/tet-DNA2 cells. To inhibit expression of the tet-DNA2 transgene, we added doxycycline  and confirmed no detectable Dna2 transcripts (DNA2D245A/-/tet-DNA2 (nuclease-dead) cells from the DNA2+/-/tet-DNA2 (DNA2D245A mRNA but not of wild-type DNA2 mRNA at 48 hours after doxycycline treatment (tet-DNA2 expression caused by doxycycline is hereafter shown as (-). DNA2-/-/tet-DNA2(-) and DNA2D245A/-/tet-DNA2 cells stopped proliferating  was a few times greater than in the wild-type cells  at one hour after IR . Remarkably, depletion of both Dna2 and CtIP resulted in a phenotype equivalent to that caused by the depletion of either single gene, in terms of both percentage of Rad51-foci-positive cells and the number of Rad51 foci  mutation knockin construct. The gene-disruption construct was designed for deletion of exons 6 to 12. The D245A mutation was integrated into exon 5. The boxes represent exons. The relevant BamHI and StuI cleavage sites are indicated. BamHI site (indicated by dotted line with an arrowhead) was integrated with the marker. (B) Southern blot analysis of wild-type (WT), DNA2+/-/tet-DNA2, and DNA2-/-/tet-DNA2 cells using the probe (bold bars) shown in A. Genomic DNA was digested with BamHI and StuI. (C) RT-PCR using the primers shown in A was done 48 hours after addition of doxycycline (Dox). (D and E) Southern blot and RT-PCR were, respectively, carried out for the indicated genotypes. The RT-PCR product amplified from the D245A mutant mRNA, but not wild-type mRNA, was digested with BstUI. The silent mutation that generates BstUI site was introduced nearby D245A mutation.(A) Schematic representation of the (EPS)Click here for additional data file.S2 Fig1 cells, the upper-middle box identifies S cells, and the lower-right box identifies G2 cells. (B) Summary of cell cycle analysis. (C) Representative chromosomal aberrations in chromosome spreads of mitotic cells. Orange, green, and black arrowheads represent chromatid-type, isochromatid-type breaks, and exchanged chromosomes, respectively. (D) Recombinant proteins used in (A) Cell-cycle distribution of the indicated genotypes. Cells were continuously treated with (+) or without (-) doxycycline (100 ng/ml) for 96 hours. Cells were stained with FITC-conjugated anti-BrdU antibody to measure BrdU incorporation into genomic DNA  and with propidium iodide (PI) to measure the total DNA . The large box on the left identifies sub G1 fraction, the lower-middle box identifies G(EPS)Click here for additional data file.S3 Fig2 phases by knockdown of the Dna2 protein. PI staining is shown on the x-axis  in both histogram and dot plots. The y-axis of dot plots is shown as in 2 phases. (C) Representative images merged with Dna2 and γ-H2AX foci of DT40 cells at 30 minutes post-IR. Green, red, and blue indicate γ-H2AX foci, Dna2 foci and DNA, respectively. (D) Representative images of RPA foci in human U2OS cells treated with the indicated siRNAs. (E) Kinetics of Dna2-focus formation in both Hela and U2OS cell lines after IR treatment.(A) Representative images of Rad51 foci (green) in the indicated chicken DT40 genotypes at 1 hour after IR. (B) Accumulation of human U2OS cells in late-S and G(EPS)Click here for additional data file.S4 FigCtIP locus and configuration of gene-disruption with N183AR187A (nuclease-dead) mutation knockin construct. The gene-disruption construct was designed for deletion of exons 6 to 10. The N183A, R187A (nuclease-dead) mutation was integrated into exon 7. The boxes represent exons. (B) The flow chart describes the generation process of CtIPN183AR187A/-/-, tetCtIP from CtIP+/-/-, tetCtIP cells. (C) RT-PCR using a couple of primers flanking the mutation region was done 96 hours after addition of doxycycline (Dox). The RT-PCR product amplified from the nuclease-dead mutant (CtIPN183AR187A /-/-) mRNA, but not wild-type mRNA, was digested with BfaI. The silent mutation that generates BfaI site was introduced nearby N183AR187A mutation.  The DT40 nuclease-dead CtIP cells are sensitive to epotoside (D) and campothecin (E), but not to IR (F). The survival curves were plotted using colony formation assays. (G) Representative images of Rad51 foci (green) (upper) and the average number of Rad51 foci per cell in indicated genotypes at one hour after 2 Gy γ-rays (bottom). (H) Representative images of Dna2 foci (Red) (upper) and the percentage of cells with at least four Dna2 foci in chicken DT40 clones carrying the indicated genotypes (bottom). Positive cells were counted at 30 minutes following 2 Gy γ-ray irradiation.(A) Schematic representation of the chicken DT40 (EPS)Click here for additional data file.


When we compared type I psoriasis with type II psoriasis (N = 36), we found a significant association between age at onset and the genes PSORS6, TNF-α, FCGR2A, TNFR1, CD226, HLA-C, TNFAIP3, and CCHCR1. Moreover, we replicated the association between rs12191877 (HLA-C) and type I psoriasis and between type I and type II psoriasis. Our findings highlight the role of genetics in age of onset of psoriasis.Psoriasis is a chronic skin disease in which genetics play a major role. Although many genome-wide association studies have been performed in psoriasis, knowledge of the age at onset remains limited. Therefore, we analyzed 173 single-nucleotide polymorphisms in genes associated with psoriasis and other autoimmune diseases in patients with moderate-to-severe plaque psoriasis type I  or type II  and healthy controls. Moreover, we performed a comparison between patients with type I psoriasis and patients with type II psoriasis. Our comparison of a stratified population with type I psoriasis ( IL23R) and IL12B and tumor necrosis factor alpha (TNFα) are closely associated with psoriasis and related diseases such as rheumatoid arthritis, psoriatic arthritis, and Crohn's disease [∗0602 is the allele most closely associated with this disease [Psoriasis is a chronic inflammatory skin disorder with a major genetic component. The prevalence of chronic plaque psoriasis is around 2% in the general population . The man∗0602 is more strongly associated with type I psoriasis than with type II psoriasis [The age at onset of psoriasis follows a bimodal distribution : type I soriasis . AlthougWe recruited 198 Caucasian patients with moderate-to-severe plaque type psoriasis (psoriasis area and severity index > 10) who attended the department of dermatology in four university hospitals in Madrid between 16/10/2007 and 17/12/2012. Five samples did not fulfill the quality criteria of the Human Genotyping Unit-CeGen , and 2 samples had insufficient volume. We also included 197 healthy volunteers (controls) recruited between 10/01/2011 and 14/12/2012 from the Clinical Pharmacology Service . All the volunteers were Caucasian and had no personal or family history of psoriasis (at least 2 generations).The protocol fulfilled Spanish law on biomedical research and was approved by the Ethics Committee for Clinical Investigation of Hospital Universitario de la Princesa. All controls and patients gave their written informed consent to donate a sample for investigation. The samples are kept in the Clinical Pharmacology Service.We preselected 320 SNPs based on an extensive review of 449 articles describing the association between polymorphisms and psoriasis and response to biological drugs and psoriasis and related inflammatory diseases  [A 3-mL peripheral blood sample was extracted from each subject in EDTA tubes. DNA was obtained from samples using an automatic DNA extractor  and its concentration was quantified in Nanodrop ND-1000 Spectrophotometer . The extracted DNA was stored at −80°C in the Clinical Pharmacology Service until use.A total of 196 samples from patients (2 samples of 198 cases had insufficient volume) and 197 samples from controls were sent to the Human Genotyping Unit-CeGen to genotype 192 SNPs. The analysis was performed using the Illumina Veracode genotyping platform. If fluorescence was low or the genotype clusters were undifferentiated, the SNPs were removed. In addition, if the call rate was less than 95% of the average of the 192 SNPs analyzed, the samples were removed. Since CEGEN quality criteria were not met in 19 SNPs and 5 patients, we finally analyzed 173 SNPs in 191 patients and 197 controls.N = 155) or type II psoriasis (N = 36) versus controls (N = 197) and patients with type I psoriasis versus cases with type II psoriasis. Hardy-Weinberg equilibrium was tested for all the SNPs analyzed using the SNPStats program [The statistical analysis was performed to compare the following stratified populations: patients with type I psoriasis  [p < 0.1 in the univariate analysis (adjusted for rs12191877) were included in a multivariate logistic regression model to adjust for relevant confounding factors (SPSS 15.0). The results of the univariate analysis were adjusted for rs12191877, except when we compared patients with type I psoriasis and patients with type II psoriasis (the influence of rs12191877 was not very relevant). We expressed the results as the odds ratio (OR), 95% confidence interval, and p value.The univariate analysis was performed using R 3.0.2. (SNPassoc) . We consulation) . The optThe study population included 155 patients with moderate-to-severe chronic plaque type I psoriasis (92 men and 63 women), 36 patients with type II psoriasis (19 men and 17 women), and 197 controls (98 men and 99 women). The mean age was 46.01 ± 13.11 years in patients with type I psoriasis (45.72 ± 11.69 in men and 46.43 ± 15.04 in women), 67.72 ± 11.85 years in patients with type II psoriasis (65.95 ± 11.18 in men and 69.71 ± 12.59 in women), and 24.51 ± 4.29 years in the controls (25.07 ± 4.94 in men and 23.95 ± 3.46 in women). The mean age at onset of psoriasis was 23.31 ± 8.52 in patients with type I psoriasis and 52.58 ± 10.45 in patients with type II psoriasis. Analysis of the effect of sex on our results revealed no significant association. JUNB gene) and was excluded from the statistical analysis. The genotyping success rate was 89.82%, and the reproducibility rate was 100%.A total of 192 SNPs were analyzed (see supplementary Table S1 published in ). HoweveAll the minor allele frequencies were in Hardy-Weinberg equilibrium except 9 SNPs in the controls and 12 SNPs in the patients (see supplementary Table S1 published in ). The 9 N = 155 versus N = 197 controls): rs1634517 (CCL4L), rs1975974 (C17orf51), rs12720356 (TYK2), rs1800925 (IL13), and rs6908425 (CDKAL1) decreased the risk of psoriasis 2.94-fold, 2.08-fold, 10-fold, 100-fold, and 2.44-fold, respectively; and rs2282276 (CLMN), rs10782001 (FBXL19), rs3792876 (SLC22A4), rs12191877 (HLA-C), and rs13437088 (HLA-B/MICA) increased the risk of psoriasis 3.90-fold, 2.10-fold, 3.75-fold, 30.54-fold, and 2.52-fold, respectively (Our findings showed an association between type I psoriasis and 10 SNPs (ectively . HoweverN = 155) compared with type II psoriasis (N = 36), namely, rs191190 , rs361525 , and rs10499194 and rs6920220 . We also found 5 SNPs that were associated with a significant increase in the risk of type I psoriasis, namely, rs1801274 , rs763361 , rs12459358 , rs12191877 , and rs1576  (Four SNPs were associated with significant decreases in the risk of type I psoriasis (66-fold) .About 75% of patients with chronic plaque psoriasis have type I psoriasis before age 40 . Our res CLMN, FBXL19, CCL4L, C17orf51, TYK2, IL13, SLC22A4, CDKAL1, HLA-C, and HLA-B/MICA.When we compared patients with type I psoriasis and controls, we found 10 significant SNPs in∗0602 allele is a risk factor for psoriasis [HLA-C) and type I psoriasis compared with type II psoriasis (OR = 3.24 in type I) [ HLA-C gene was associated with type I psoriasis , but not with type II psoriasis after application of the Bonferroni correction [HLA-C) in patients with type I psoriasis (p = 2.50E − 19). However, we did not find this association in patients with type II psoriasis, probably owing to the small sample size in this group (N = 36).The HLA-Csoriasis  and has soriasis  and typesoriasis . In one soriasis , whereassoriasis . Other a type I) . rs10484rrection . In addi IL13 gene and type I psoriasis (p = 2.47E − 03) [ IL13 (rs1800925) and type I psoriasis (p = 0.034). In addition, Munir et al. did not obtain significant results when they compared controls with type II psoriasis or type I psoriasis with type II psoriasis [ IL13 have been associated with predisposition to psoriasis [Munir et al. found an association between rs1295685 in the7E − 03) . Our ressoriasis . Both SNsoriasis , 37. FBXL19 [ C17orf51 [ TYK2 [ SLC22A4 [ CDKAL1 [ HLA-B/MICA [Our comparison of patients with type I psoriasis and controls is the first to obtain significant results for a series of SNPs in type I psoriasis, although the SNPs have already been associated with the risk of psoriasis. rs10782001 in FBXL19 , rs19759C17orf51 , rs127201 [ TYK2 , 39, rs3[ CDKAL1 , and rs1A-B/MICA  have pre CLMN (rs2282276) and CCL4L (rs1634517) have not been associated with psoriasis or age at onset.Furthermore, SNPs inN = 36).We found no significant differences between patients with type II psoriasis and controls owing to the small sample size  have been associated with type I psoriasis [ CCHCR1 and age at onset. In a study comparing controls (54.8%) and patients with psoriasis type II (66.0%), Allen et al. showed a significant increase in the number of patients carrying rs1576 [Polymorphisms insoriasis , 31. We g rs1576 . This SNg rs1576 . CD226 in patients with early-onset psoriasis and patients with late-onset psoriasis, although they found no associations [ CD226 has been associated with severity of psoriasis [Douroudis et al. analyzed rs763361 inciations . We perfsoriasis . PSORS6 has been associated with type I psoriasis  [p = 0.026).rs12459358 in= 0.005) . In cont TNFα gene has been associated with susceptibility to psoriasis [p = 2E − 07) [TNF-α) when we compared patients with type I psoriasis and patients with type II psoriasis, although we found no gender differences. Other authors confirmed our association with type I psoriasis in Caucasian [TNFα) and type I or type II psoriasis [rs361525 −238) in thesoriasis , and the2E − 07) . We founaucasian , 23 and 8 in thesaucasian . A meta-aucasian . Baran esoriasis . HLA-C has been associated with increased risk of psoriasis [ HLA-C. We replicated these results in rs12191877 .Likewise, rs12191877 insoriasis . Munir esoriasis  compared TNFR1 [ TNFAIP3 [ FCGR2A and rs6920220 in TNFAIP3 have not been studied in patients with psoriasis according to age of onset. Given the small sample size in the group with type II psoriasis in our study, our results should be interpreted with caution.rs191190 in TNFR1  and rs10Our results highlight the role of the immune system in psoriasis and enhance our understanding of pathogenic mechanisms. Such knowledge can help to optimize treatment.Our study is subject to a series of limitations. First, mean age varied between the cases and the controls. Second, the sample size was limited by the number of study patients treated in the dermatology department, thus making it difficult to detect SNPs with a low probability of causing psoriasis. Third, since the SNPs were selected based on a literature review, several major SNPs may not yet have been investigated.HLA-C) and type I psoriasis and between type I and type II psoriasis patients. Ours is the first study to show an association between CLMN, FBXL19, CCL4L, C17orf51, TYK2, IL13, SLC22A4, CDKAL1, and HLA-B/MICA and type I psoriasis. Moreover, CLMN and CCL4L have not been previously described in psoriasis. In addition, PSORS6 and TNFα have been described as more prevalent genes in type I psoriasis and we showed a significant association when we compared type I psoriasis and type II psoriasis. Ours is the first study to identify an association between FCGR2A, TNFR1, CD226, TNFAIP3, and CCHCR1 and age at onset of psoriasis. Our results suggest that genetics could play a role in age at onset. However, further studies are needed to confirm our findings.In conclusion, our study confirmed an association between rs12191877 (


Recurrence or early metastasis remains the predominant cause of mortality in patients with estrogen receptor positive (ER+) mammary carcinoma (MC). However, the molecular mechanisms underlying the initial progression of ER+ MC to metastasis remains poorly understood. Trefoil factor 3 (TFF3) is an estrogen-responsive oncogene in MC. Herein, we provide evidence for a functional role of TFF3 in metastatic progression of ER+ MC.in vitro and in vivo. Mechanistic analyses were performed using reporter constructs, modulation of signal transducer and activator of transcription 3 (STAT3) expression, and pharmacological inhibitors against c-SRC and STAT3 activity.The association of TFF3 expression with clinicopathological parameters and survival outcome in a cohort of MC patients was assessed by immunohistochemistry. The expression of TFF3 in MCF7 and T47D cells was modulated by forced expression or siRNA-mediated depletion of TFF3. mRNA and protein levels were determined using qPCR and western blot. The functional effect of modulation of TFF3 expression in MC cells was determined In vivo, forced expression of TFF3 in MCF7 cells stimulated the formation of metastatic nodules in animal lungs. TFF3 regulation of the mRNA levels of epithelial, mesenchymal, and metastatic-related genes in ER+ MC cells were consistent with the altered cell behaviour. Forced expression of TFF3 in ER+ MC cells stimulated phosphorylation of c-SRC that subsequently increased STAT3 activity, which lead to the downregulation of E-cadherin. siRNA-mediated depletion of TFF3 reduced the invasiveness of ER+ MC cells.TFF3 protein expression was positively associated with larger tumour size, lymph node metastasis, higher stage, and poor survival outcome. Forced expression of TFF3 in ER+ MC cells stimulated colony scattering, cell adhesion to a Collagen I-coated matrix, colony formation on a Collagen I- or Matrigel-coated matrix, endothelial cell adhesion, and transmigration through an endothelial cell barrier. TFF3 expression predicts metastasis and poor survival outcome of patients with MC and functionally stimulates cellular invasion and metastasis of ER+ MC cells. Adjuvant functional inhibition of TFF3 may therefore be considered to ameliorate outcome of ER+ MC patients.The online version of this article (doi:10.1186/s13058-014-0429-3) contains supplementary material, which is available to authorized users. Metastatic sequelae are the predominant causes of mortality in patients with mammary carcinoma (MC) . The metTFF3 mRNA predicts a worse survival outcome in ER+ patients treated with tamoxifen and increased expression of TFF3 both reduces sensitivity of ER+ MC cells to tamoxifen and mediates acquired resistance to tamoxifen . El. El11]. amoxifen . Inhibitamoxifen . A numbeamoxifen ; TFF3 examoxifen ,17]; TF; TFTFF3 amoxifen . A recenamoxifen , the funin vitro and in vivo, and dependent on v-src avian sarcoma (Schmidt-Ruppin A-2) viral oncogene homologue-signal transducer and activator of transcription 3 (c-SRC- STAT3)-mediated repression of CDH1. Thus, functional inhibition of TFF3 may be considered as an adjuvant intervention to reduce metastatic seeding and ameliorate disease outcomes in patients with ER+ MC.In this study, we sought to determine if TFF3 is functionally associated with increased metastatic potential of ER+ MC cells. We first observed that TFF3 protein expressed in MC specimens was positively associated with metastasis and poor survival outcomes of patients with MC. Forced expression of TFF3 in ER+ MC cells was associated with increased invasion and metastatic seeding, as assessed The human ER+ MC cell lines, MCF7 and T47D, were obtained from the American Type Culture Collection  and were cultured as per ATCC propagation instructions. Stable cell lines were freshly generated as previously described . STAT3 aP <0.05) with both increased tumour size and lymph node metastases.The patient cohort used herein consists of 159 MC and 33 specimens of benign mammary disease (BMD) that underwent surgery at the First Affiliated Hospital of Anhui Medical University (AMU)  between 2001 and 2002 as previously described . The InsTFF3 expression and siRNA plasmid constructs have been previously described . Co. Co32]. in vitro invasiveness of MC cells . We. Wein viDuring the process of metastasis, carcinoma cells adhere to and migrate through endothelial cells that line the blood vessels in a process known as transendothelial migration ,35]. We. We35]. To determine whether TFF3 contributes to the metastatic seeding of MC cells, MCF7-vector and MCF-TFF3 cells were injected into the tail vein of BALB/c nude mice (n = 6 each group) and examined their ability to form metastatic nodules Figure A. HistolhHPRT) mRNA in both organs. We observed that the level of hHPRT mRNA expression was increased >100-fold in the lungs of animals injected with MCF7-TFF3 cells compared with MCF7-vector cells. Minimal hHPRT mRNA was detected in the liver of mice injected with MCF-vector cells and no significant differences of hHPRT mRNA expression were observed in liver tissue from mice injected with either MCF7-vector or MCF7-TFF3 cells. Mouse glyceraldehyde 3-phosphate dehydrogenase (mgapdh) was used as an internal control  of STAT3 in colon carcinoma cell lines --[17-[1715]- and andTFF3 amoxifen ,[15], and andTFF3 amoxifen . It is ramoxifen , ER posiamoxifen . It was amoxifen . In the amoxifen ,51] and andTFF3 amoxifen . Hence, amoxifen , yet BCLamoxifen ,13]. It. ItTFF3 amoxifen ), the maamoxifen  and consβ and/or EGF/EGFR-mediated STAT3 activation also has been observed to possess a pivotal role in EMT through increased TWIST1 expression in MC cells . Pe. Pe57]. MC cells . TWIST2 MC cells . ConcordMC cells . Indeed,MC cells . Such a MC cells . Herein,MC cells . Enhanceamoxifen . MCF7 ceamoxifen . An incramoxifen . Concord protein . Additio protein . Our curin vivo dissemination of MC cells . Fu. Fuin viMC cells . FunctioMC cells . We haveMC cells , SNAIL aMC cells . An incrMC cells . MoreoveMC cells . In a lah ER+ MC . We haveh ER+ MC . Elevateh ER+ MC . Although ER+ MC ; we haveh ER+ MC . Future In summary, we have demonstrated that TFF3 functionally promotes metastatic seeding of ER+ MC cells. Forced expression of TFF3 in ER+ MC cells promotes a mesenchymal and invasive phenotype. TFF3 stimulated an invasive phenotype in ER+ MC cell through c-SRC-STAT3 mediated repression of CDH1. Furthermore, TFF3 expression predicts metastasis and poor survival outcome of patients with MC. The current findings are therapeutically relevant as early metastatic dissemination and late relapse represents a significant threat to long-term survival of patients with ER+ MC. Inhibition of TFF3 function in MC warrants consideration.PEL designed the research, analyzed the data, and wrote the manuscript. VP designed the research, conducted the experiments, analyzed the data, and wrote the manuscript. ZSW conducted the experiments and analyzed the data. JZ, RL, and MZ conducted the experiments and TZ analyzed the data. All authors have read and approved the manuscript for publication.Additional file 1: (A) Histopathological scoring. (B) qPCR primer sequence.(PDF 96 KB)Forced expression of TFF3 in MC cells stimulates invasion.(A) mRNA levels of TFF3 in various MC cell lines derived from the Oncomine database (http://www.oncomine.org) and previously reported [(B and C) Migration and invasion of MCF7 and T47D cells, with either forced or depleted expression of TFF3, was determined by Transwell chamber assay. (D) Wound-healing assay, wounded areas were examined under X100 magnification using a phase contrast microscope. (E) Invasive capacity of MCF7 and T47D cells with either forced or depleted expression of TFF3 on exposure to vehicle (DMSO) and/or transiently transfected with control plasmids, was determined by Transwell chamber assay. Statistical significance was assessed by using an unpaired two-tailed Student's t test (P <0.05 was considered as significant) using GraphPad Prism 5. Columns are the mean of triplicate experiments; bars, ± SD. **P <0.001, *P <0.05. (PDF 367 KB)Additional file 2: reported . Cell liForced expression of TFF3 in T47D cells modulates the mRNA levels of epithelial, mesenchymal and metastatic-related gene markers.(A) qPCR analyses of T47D cells with either forced or depleted expression of TFF3 for mRNA levels of key genes functionally involved in migration, invasion, metastasis, and EMT. Change in gene expression is expressed as fold difference, respectively. Fold change values are representative of three independent biological experiments. Statistical significance was assessed by using an unpaired two-tailed Student's t test (P <0.05 was considered as significant) using GraphPad Prism 5. (B) Western blot analysis was used to assess the protein levels of epithelial and mesenchymal markers in T47D cells with either forced or depleted expression of TFF3 as described in Methods. (C) Confocal microscopic visualisation of CDH1 expression in MCF7 and T47D cells with forced expression of TFF3 after exposure to JSI-124 (0.2 μM) or Stattic (2 μM). The white colour indicates CDH1 expression, and blue colour indicates nuclei stained with DAPI. Images were captured under oil immersion X600 magnification. (D) Confocal microscopic visualisation of VIM expression in T47D cells with either forced or depleted expression of TFF3. The red colour indicates VIM expression, and blue colour indicates nuclei stained with DAPI. Images were captured under oil immersion X600 magnification. (E). Visualization of CDH1 expression in T47D cells with siRNA-mediated depleted expression of TFF3. The white colour indicates CDH1 expression, and blue colour indicates nuclei stained with DAPI. Images were captured under oil immersion X600 magnification. (PDF 430 KB)Additional file 3: Forced expression of TFF3 in T47D cells enhanced invasive phenotype.(A) Confocal microscopic visualisation of f-actin arrangement in T47D cells with either forced or depleted expression of TFF3. The red colour indicates f-actin. Images were captured under X200 magnification. (B) Distribution of compact, loose, and scattered colonies of T47D cells with either forced or depleted expression of TFF3 as described in Methods. Right side, illustrative images of compact, loose, and scattered monolayer adherent colonies of T47D, with either forced or depleted expression of TFF3. (C) Capacity of T47D cells with either forced or depleted expression of TFF3 to adhere to a Collagen I matrix. (D) Morphology of T47D cells with either forced or depleted expression of TFF3 when cultured on a Collagen I matrix. Statistical significance was assessed by using an unpaired two-tailed Student's t test (P <0.05 was considered as significant) using GraphPad Prism 5. Columns or points are the mean of triplicate experiments; bars, ± SD. **P <0.001, *P <0.05. (PDF 193 KB)Additional file 4: Forced expression of TFF3 in MC cells stimulated growth on two dimensional Matrigel; and forced expression of TFF3 in T47D cells enhanced adherence to collagen I and endothelial cells; and transmigration through an endothelial cell layer.(A) Cell viability and morphology (below) of MCF7 (left side) and T47D (right side) cells with either forced or depleted expression of TFF3 when cultured on Matrigel-coated  plates. Images were captured under X200 magnification using phase-contrast microscopy. (B) Capacity of T47D cells with either forced or depleted expression of TFF3 to adhere to HMEC-1 cells. The green colour indicates T47D cells (either forced or depleted expression of TFF3), and the red colour indicates HMEC-1 cells (down side). Images were captured under X100 magnification using a fluorescence microscope, as described in Methods. (C). Capacity of T47D cells with either forced or depleted expression of TFF3 to transmigrate through a HMEC-1 monolayer, as described in Methods. Statistical significance was assessed by using an unpaired two-tailed Student's t test (P <0.05 was considered as significant) using GraphPad Prism 5. Columns or points are the mean of triplicate experiments; bars, ± SD. **P <0.001, *P <0.05. (PDF 208 KB)Additional file 5: Repression of CDH1 expression is required for TFF3-stimulated invasion of MC cells.(A) Effect of forced expression of CDH1 on the MCF7 and T47D cell invasion with either forced or depleted expression of TFF3 was evaluated using a Transwell assay. Statistical significance was assessed by using an unpaired two-tailed Student's t test (P <0.05 was considered as significant) using GraphPad Prism 5. Columns are the mean of triplicate experiments; bars, ± SD. **P <0.001, *P <0.05. (PDF 10 KB)Additional file 6: Forced expression of TFF3 in T47D cells stimulated phosphorylation of STAT3 to promote invasion.(A) Western blot analysis was used to assess the levels of pSTAT3, and STAT3 in T47D cells with either forced or depleted expression of TFF3 as described in Methods. (B) STAT3 mediated transcription, α2-M promoter activity in T47D cells with either forced or depleted expression of TFF3 on exposure to JSI-124 (0.2 μM) or Stattic (2 μM); and/or transiently transfected with STAT3 DN, STAT3-siRNA or STAT3 CA. The luciferase assay was performed as described in Methods. (C)CDH1 promoter activity in T47D cells with either forced or depleted expression of TFF3 on exposure to JSI-124 (0.2 μM) or Stattic (2 μM). The luciferase assay was performed as described in Methods. (D) Western blot analysis was used to assess the levels of CDH1 in T47D cells with forced expression of TFF3 on exposure to JSI-124 (0.2 μM) or Stattic (2 μM) inhibitor as described in Methods. (E) Invasive capacity of T47D cells with either forced or depleted expression of TFF3 on exposure to JSI-124 (0.2 μM) or Stattic (2 μM); and/or transiently transfected STAT3 DN, STAT3-siRNA or STAT3 CA. Cell invasion was evaluated using a Transwell assay. Statistical significance was assessed by using an unpaired two-tailed Student's t test (P <0.05 was considered as significant) using GraphPad Prism 5. Columns are the mean of triplicate experiments; bars, ± SD. **P <0.001, *P <0.05. (PDF 74 KB)Additional file 7: Forced expression of TFF3 in T47D cells stimulated phosphorylation of c-SRC that subsequently increased STAT3 activity to promote invasion.(A) Western blot analysis was used to assess the levels of p-c-SRC and c-SRC in T47D cells with either forced or depleted expression of TFF3. (B) Western blot analysis was used to assess the protein levels of pSTAT3, STAT3, and CDH1 in T47D cells with forced expression of TFF3 on exposure to PP1 (5 μM), PP2 (2 μM) or PP3 (50 μM as described in Methods. (C) CDH1 promoter activity in T47D cells with either forced or depleted expression of TFF3, on exposure to PP1 (5 μM), PP2 (2 μM) or PP3 (50 μM). The luciferase assay was performed as described in Methods. (D) STAT3 mediated transcription, α2-M promoter activity in T47D cells with either forced or depleted expression of TFF3, on exposure to PP1 (5 μM), PP2 (2 μM) or PP3 (50 μM). The luciferase assay was performed as described in Methods. (E) Invasive capacity of T47D cells with either forced or depleted expression of TFF3, on exposure to PP1 (5 μM), PP2 (2 μM) or PP3 (50 μM); and/or in combination with transiently transfected STAT3 CA. Invasion was evaluated using a Transwell assay. Statistical significance was assessed by using an unpaired two-tailed Student's t test (P <0.05 was considered as significant) using GraphPad Prism 5. Columns are the mean of triplicate experiments; bars, ± SD. **P <0.001, *P <0.05. (PDF 116 KB)Additional file 8: Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Authors’ original file for figure 4Authors’ original file for figure 5Authors’ original file for figure 6Authors’ original file for figure 7Below are the links to the authors’ original submitted files for images.