CUL4A has been proposed as oncogene in several types of human cancer, but its clinical significance and functional role in human non-small cell lung cancer (NSCLC) remain unclear.in vitro and tumorigenesis in vivo, respectively.Expression level of CUL4A was examined by RT-PCR and Western blot. Forced expression of CUL4A was mediated by retroviruses, and CUL4A silencing by shRNAs expressing lentiviruses. Growth capacity of lung cancer cells was measured by MTT We found that CUL4A was highly expressed in human lung cancer tissues and lung cancer cell lines, and this elevated expression positively correlated with disease progression and prognosis. Overexpression of CUL4A in human lung cancer cell lines increased cell proliferation, inhibited apoptosis, and subsequently conferred resistance to chemotherapy. On other hand, silencing CUL4A expression in NSCLC cells reduced proliferation, promoted apoptosis and resulted in tumor growth inhibition in cancer xenograft model. Mechanistically, we revealed CUL4A regulated EGFR transcriptional expression and activation, and subsequently activated AKT. Targeted inhibition of EGFR activity blocked these CUL4A induced oncogenic activities.Our results highlight the significance of CUL4A in NSCLC and suggest that CUL4A could be a promising therapy target and a potential biomarker for prognosis and EGFR target therapy in NSCLC patients.The online version of this article (doi:10.1186/1476-4598-13-252) contains supplementary material, which is available to authorized users. Lung cancer remains by far the most common cause of cancer mortality and non-small cell lung cancer (NSCLC) accounts for >80% of cases of lung cancer, which ranks among the most deadly cancers worldwide. AlthougEpidermal growth factor receptor (EGFR) is a transmembrane protein with intrinsic tyrosine kinase activity that regulates cell growth in response to binding of its ligands. EGFR is overexpressed or mutated in most NSCLC cases, and deregulated expression of EGFR together with ligand binding and concomitant receptor activation promotes tumor cell growth, proliferation, and survival, 4. Seveet al. reported that DLC1 was ubiquitinated and degraded by cullin 4A-RING ubiquitin ligase (CRL4A) complex interaction with DDB1 and the FBXW5 substrate receptor in NSCLC cell lines[CUL4A, a member of the cullin family of proteins that composes the multifunctional ubiquitin ligase E3 complex, plays critical roles in DNA replication, cell cycle regulation and genomic instability–15. CUL4ll lines. The recll lines. HoweverIn the present work, we sought to investigate the role and mechanism of CUL4A in NSCLC. We first examined both mRNA and protein expression patterns and evaluated prognostic significance of CUL4A in NSCLC. High levels of CUL4A predicted poor prognosis in overall survivals. Moreover, ectopic expression of CUL4A promoted cell proliferation and inhibited apoptosis. Knockdown of endogenous CUL4A by shRNA significantly decreased cell proliferation and tumorigenesis. Those oncogenic functions of CUL4A are at least partially mediated by regulation of EGFR and its related pathways. Additionally, we showed that CUL4A overexpression conferred NSCLC cells resistance to chemotherapy and sensitivity to EGFR target therapy. Our findings implicate CUL4A as a promising molecular target for therapy and a prognostic marker for highly recurrent NSCLC.P <0.001, Figure We first examined CUL4A expression in a panel of 7 human lung cancer cell lines and 2 normal human lung epithelial cell lines. RT-PCR  and silenced CUL4A expression (designated as A549-shCUL4A and H460-shCUL4A) were verified by RT-PCR  , for 4 h. When EGFR phosphorylation was blocked by erlotinib, CUL4A induced AKT phosphorylation was reduced Figure C.Figure To determine if the proliferative effect of CUL4A on NSCLC cells was EGFR dependent, we treated H1299-CUL4A, H1650-CUL4A and their control cells with erlotinib. Erlotinib clearly reduced the promotive effect of CUL4A on cell proliferation , II (n =20), III (n =25), and IV (n =16). A total of 22 fresh tumor tissues and 22 fresh normal lung tissues were stored at -70°C immediately after resection for extraction of RNA.BEAS2B, HSAEpiC, A549, H1299, H460, A427, H1650, 95D, and HLAMP cell lines were from American Type Culture Collection . The cells were cultured in RPMI 1640  containing 10% fetal calf serum (Invitrogen), 100 IU/ml penicillin , and 100 μg/ml streptomycin (Sigma). Cells were grown on sterilized culture dishes and were passaged every 2 days with 0.25% trypsin (Invitrogen).CUL4A cDNA and pSuper.retro.puro with shRNA against human CUL4A cDNA were prepared as described previously[pBabe-puro retroviral constructs containing human eviously. The conImmunostaining was performed using the avidin-biotin-peroxidase complex method . The sections were deparaffinized in xylene, rehydrated with graded alcohol, and then boiled in 0.01 M citrate buffer (pH 6.0) for 2 min with an autoclave. Hydrogen peroxide (0.3%) was applied to block endogenous peroxide activity, and the sections were incubated with normal goat serum to reduce nonspecific binding. Tissue sections were incubated with CUL4A rabbit polyclonal antibody (1:250 dilution), EGFR mouse monoclonal antibody (1:150 dilution). Mouse immunoglobulin (at the same concentration of the antigen specific antibody) was used as a negative control. Staining for both antibodies was performed at room temperature for 2 h. Biotinylated goat antimouse serum IgG was used as a secondary antibody. After washing, the sections were incubated with streptavidin-biotin conjugated with horseradish peroxidase, and the peroxidase reaction was developed with 3, 30-diaminobenzidine tetrahydrochloride.Two independent, blinded investigators examined all tumor slides randomly. Five views were examined per slide, and 100 cells were observed per view at 400× magnification. Scores for CUL4A and EGFR membrane and cytoplasmic staining were calculated based on staining intensity  and the percentage of cells staining at each intensity level (0-100%). The final score was calculated by multiplying the intensity score by the percentage, producing a scoring range of 0 to 300. The immunohistochemistry score cut-off point was established as 73 using X-tile software program .Total cellular RNA was extracted from cells using the RNeasy Plus Mini Kit from (Qiagen). The quality and yield of the RNA samples were determined by ultraviolet spectrophotometer. Total RNAs (1 μg) were reverse transcribed to cDNA (20 μl) using PrimeScriptTM RT Kit (TaKaRa) according to the manufacturer’s instructions. PCR reaction was conducted with 2 μL cDNA sample, 0.4 μL forward primer (10 μmol/L), 0.4 μL reverse primer (10 μmol/L), 11.2 μL RNase-free water, and 6 μL 2× EsayTaq PCR SuperMix . PCR reaction was performed using the following cycle parameters: 95°C for 5 minutes,  for 30 cycles, 72°C for 7 minutes. RT-PCR products were separated on 2% agarose gels. After stained with ethidium bromide, gel images were photographed with ChemiImagerTM 4400. RT-PCR was performed at least 3 times for each sample. The sequences of the primer pairs are:CUL4A forward, 5′ ATACTTCAGGACCCACGTTTGAT 3′,CUL4A reverse, 5′ TCTCCAAGTACTAAAGCAGGAAAATCT 3′,EGFR forward, 5′ GCCACGTCTCCACACATCAG 3′,EGFR reverse, 5′ TGGTGCATTTTCGGTTGTTG 3′,GAPDH forward, 5′ ATAGCACAGCCTGGATAGCAACGTAC- 3′,GAPDH reverse, 5′ CACCTTCTACAATGAGCT GCGTGTG 3′.GAPDH was used as the reference gene.Total protein from cells was extracted in lysis buffer (Pierce) and quantified using the Bradford method. Then, 50 μg of protein were separated by SDS-PAGE (10%). After transferring to polyvinylidene fluoride (PVDF) membrane , the membranes were incubated overnight at 4°C with antibodies against CUL4A , EGFR , β-actin . After incubation with peroxidase-coupled antimouse IgG (Santa Cruz Biotechnology) at 37°C for 2 h, bound proteins were visualized using ECL (Pierce) and detected using BioImaging Systems . The relative protein levels were calculated based on beta-actin protein as a loading control.5) were suspended in 5ml of culture medium containing 0.4% agar (USB Corportion) and seeded onto a base layer of 5ml of 0.7% agar bed in 10-cm tissure-culture dishes. Colonies >50 μm in diameter were counted after 3 weeks.The test cells . After 24 hrs, the cells were rinsed with phosphatebuffered saline (PBS) and fixed with 4% paraformaldehyde in PBS, and cell membrane was permeabilized using 0.5% Triton X-100. These cells were then blocked for 30 min in 10% BSA  in PBS and then incubated with primary monoclonal antibodies in 10% BSA overnight at 4°C. After three washes in PBS, the slides were incubated for 1 hour in the dark with FITC-conjugated secondary goat anti-mouse, or goat anti-rabbit antibodies . After three further washes, the slides were stained with 4-,6-diamidino-2-phenylindole  for 5 min to visualize the nuclei, and examined using an Carl Zeiss confocal imaging system (LSM 780) .Cells were plated in 96-well plates in medium containing 10% FBS at about 3,000 cells per well 24 h after transfection. Then, 20 μl of 5 mg/ml MTT (Thiazolyl Blue) solution was added to each well and incubated for 4 h at 37°C, the media was removed from each well, and the resultant MTT formazan was solubilized in 150 μl of DMSO. The results were quantitated spectrophotometrically using a test wavelength of 570 nm.5/mL. In the dark, 5 μL Annexin V-FITC  was added to cell suspension Mix of 195 μL and incubated for 10 min at room temperature before adding 190 μL Binding buffer (1×) and 10μL PI. Ten thousand events per sample were acquired using a FACS-scan flow cytometer  and the percentage of cell apoptosis were analyzed using Cell Quest analysis software (Becton-Dickinson).Cells were harvested and washed twice with cold PBS by gentle shaking. Resuspend cells were added to Binding buffer and adjusted cell density to 2–5 × 10Cells were fixed in 1% formaldehyde for 10 minutes at 37°C. Cross-linking was quenched by adding 125 mmol/L glycine. Cells were then washed with cold PBS, harvested and resuspended in SDS lysis buffer containing a protease inhibitor cocktail. Chromatin was sheared by sonication (average length 0.25-1 Kb) and incubated with 60 ml protein A/G agarose/salmon sperm DNA  with gentle agitation for 30 minutes. The supernatant was then immunoprecipitated with anti-SOX4 antibody 1:500 or its matched nonimmune crude serum 1:500  at 4°C overnight. Protein A/G agarose (60 mL of 50% slurry) was then added and incubated for 1 hour. Pellets were washed and protein-DNA cross-links were reversed by overnight incubation at 65°C with proteinase K. DNA was purified following a conventional phenol–chloroform protocol and eluted in 50 mL water. At least 3 independent Chromatin immunoprecipitation (ChIP) experiments were carried out.6, suspended in 100 μL sterile PBS) per mouse in the right oxter as control group. The other group was inoculated with A549/CUL4A shRNA cells . Tumor volume was calculated using the equation (L × W2)/2.Female BALB/c nude mice  were purchased from the Center of Experimental Animal of Guangzhou University of Chinese Medicine and were housed in barrier facilities on a 12-hour light/dark cycle. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Shandong University. The BALB/c nude mice were randomly divided into 2 groups (n =6/group). One group of mice were inoculated subcutaneously with A549/vector cells  RT-PCR analysis of CUL4A mRNA levels in nine lung cell lines. (B) Western blot analysis of CUL4A protein levels in lung cancer cell lines. All experiments were repeated three times. Error bar indicate standard deviation. (JPEG 2 MB)in vitro. Cell proliferation in vitro was examined by MTT in H1650-pbabe, H1650-CUL4A (A) and H460-pSuper, H460-shCUL4A (B) cells. (JPEG 781 KB)Additional file 2: Figure S2: CUL4A regulates NSCLC cell growth both in vitro. (A) Photomicrographs illustrating examples of soft agar colonies (left) and histobars indicating the statistical significance of the numbers of colonies (right) in H1299-pBabe and H1299-CUL4A cells. (B) Photomicrographs illustrating examples of soft agar colonies (left) and histobars indicating the statistical significance of the numbers of colonies (right) in A549-pSuper and A549-shCUL4A cells. **P <0.01. (JPEG 984 KB)Additional file 3: Figure S3: CUL4A-induced lung cancer cell transformation Additional file 4: Figure S4: The immunohistochemistry analysis of Ki67 expression in CUL4A-pBabe and CUL4A-shCUL4A cells xenograft tumors. Scale bar indicates 50 μm. (JPEG 777 KB)*P <0.05 and **P <0.01 vs pBabe cells; #P <0.05 and ##P <0.01 vs pSuper cells. All results are from three independent experiments. Error bar indicate standard deviation. (JPEG 2 MB)Additional file 5: Figure S5: CUL4A regulated the sensitivity of NSCLC cells to chemotherapy. (A) MTT analysis of the viability of H1299 cell treated with different doses of doctaxel. (B) MTT analysis of the viability of H1299 cell treated with different doses of doxorubicin. (C) MTT analysis of the viability of H1650 cell treated with different doses of doctaxel. (D) MTT analysis of the viability of H1650 cell treated with different doses of doxorubicin. (E) MTT analysis of the viability of A549 cell treated with different doses of doctaxel. (F) MTT analysis of the viability of A549 cell treated with different doses of doxorubicin. (G) MTT analysis of the viability of H460 cell treated with different doses of doctaxel. (H) MTT analysis of the viability of H460 cell treated with different doses of doxorubicin. Additional file 6: Figure S6: The immunohistochemistry analysis of CUL4A and EGFR expression in CUL4A-pBabe and CUL4A-shCUL4A cells xenograft tumors. Scale bar indicates 50 μm. (JPEG 2 MB)**P <0.01 vs pBabe cells; ##P <0.01 vs CUL4A cells. All results are from three independent experiments. Error bar indicate standard deviation. (JPEG 651 KB)Additional file 7: Figure S7: LY294002 blocked the CUL4A-induced AKT phosphorylation and cell proliferation. Treatment of cells with 10 μM LY294002 blocked the induction of AKT phosphorylation (A). LY294002 also reversed proliferation of H1299 induced by CUL4A overexpression (B).


Peach allergy is prevalent, persistent and potentially severe. LTPs (Pru p 3) and profilins (Pru p 4) are the main allergens involved in this kind of allergy in patients(pts) of Mediterranean area. The hidden presence of LTPs in foodstuffs and in other food involved in LTP syndrome can trigger severe reactions, including anaphylaxis. Thus, this type of allergy can be considered an important target for specific immunotherapy(IT).To demonstrate the efficacy and safety of sublingual IT (SLIT) with Pru p 3 in pts with systemic reactions (SR) associated with peach ingestion, by evaluation of clinical and immunological parameters during 12 months.Eight pts  with a history of peach allergy, confirmed by Oral Food Challenge (unless anaphylaxis symptoms and evidence of peach sensitization), undergoing SLIT Pru p 3 (ALK-Abelló) during 1 year, were evaluated. 100% of pts had SR (75% anaphylaxis) associated with peach ingestion (62.5% reported symptoms with other foods containing LTPs). All patients underwent skin prick tests  with aeroallergens battery, peach extract peel and pulp, other foods according to symptoms, Pru p 3 and Pru p 4 (to exclude Pru p 4 sensitization). The SLIT with Pru p 3 had an induction phase , followed by outpatient maintenance phase. In all pts were performed SPT and quantified specific IgE and IgG4 (sIgE and sIgG4)-UniCAP (PhadiaThermofisher) for peach and Pru p 3 before(T0), 1(T1), 6(T6) and 12 months (T12) after SLIT initiation. Basophil Activation Test(BAT) was performed by Flow2 ® CAST method (Bühlmann) with Pru p 3 extract (Alk-Abelló) with three concentrations 0.05, 0.5 and 5ug/mL at the same times.There was a significant decrease of the mean wheal diameter of SPT in T0-T12, with peel and pulp peach (p=0.0039) and Pru p 3 (p=0.0078). Quantification of sIgE to peach and Pru p 3: significant decrease of T0-T12 (p=0.0408 and p<0.001 respectively); sIgG4: significant increase of T0-T12 (p<0.0001) for the same allergens; BAT: significant decrease between the different times and in three concentrations. During the 12 months of treatment with SLIT, there were only local reactions (itching) during the induction phase in 50% of pts with spontaneous resolution.In this initial evaluation, SLIT Pru p 3 seems to be a promising and safe therapeutic option for patients with severe peach allergy.


We showed that rice cultivars differ in aerenchyma development, nitrogen uptake and gene response to external aeration treatment. Interestingly, we found that the effect of nitrogen on rice plant growth depends on environmental O2 level.As rice adapts to waterlogged conditions it develops aerenchyma to transport O 2) level in the rhizosphere are believed to influence the behaviour of nutrients and their usage by plants. However, the effect of aeration on nitrogen (N) acquisition under different N supply conditions remains largely unknown. In this study, the rice cultivars Yangdao 6  and Nongken 57  were used to evaluate the effects of aeration on rice growth and N accumulation. Our results showed that the number of adventitious roots and the root surface area increased significantly, and ethylene production and aerenchyma formation decreased in both cultivars after external aeration (EA). Five N treatments, including no N (−N), 0.125 mM NH4NO3 (LN), 1.25 mM Ca(NO3)2 (NO3-N), 1.25 mM (NH4)2SO4 (NH4-N) and 1.25 mM NH4NO3 (N/N), were applied to YD6 and NK57 for 2 days under internal aeration or EA conditions. External aeration increased the root biomass in both cultivars and the shoot biomass in NK57 by 18–50 %. The total N concentrations in roots of YD6 grown under −N and LN and of NK57 grown under NO3-N were increased by EA. Expression of OsPAD4, one of four putative genes regulating aerenchyma formation, showed a similar pattern alongside changes in the ethylene level in the EA-treated rice irrespective of the N treatments. Furthermore, expression of the high-affinity nitrate transporter gene OsNRT2.1 was increased by EA under −N, LN and NO3-N conditions. Our data provide evidence of an interaction between O2 and the supply of N in ethylene production, aerenchyma formation and N nutrition through modification of the expression of OsPAD4 and OsNRT2.1.In wetland soils, changes in oxygen (O Both groups of rice plants were placed in a growth chamber with a 16-h/8-h light/dark cycle and 30 °C/26 °C day/night temperature cycle. Relative humidity was maintained at 60 %. After 5 days of IA or EA treatment, the numbers of adventitious roots were counted and total root lengths and root surface areas were determined  using the WinRhizoV4.0b image analysis software . Root tips were collected for aerenchyma measurements and total root systems grown under the two aeration treatments were harvested 2 h into the light phase of the daily light cycle for analysis of OsPAD4 (AK243523), OsLSD1.1 (AK111759), OsBphi008a (NM_001048814), OsPDCD5 (AY749430), OsNRT2.1 (AB008519), OsNAR2.1 (AP004023), OsNRT2.3 (AK109776), OsAMT1.1 (AF289477), OsAMT1.2 (AF289479) and OsAMT1.3 (AF289478) expression using quantitative real-time polymerase chain reaction (PCR). The shoots and roots of rice seedlings were separately collected for measuring biomass and total N. The samples were desiccated in a forced-air oven at 70 °C for ∼72 h to a constant weight, after which their dry weight was measured. The measurement of total N contents was performed according to Seeds of the YD6 and NK57 rice cultivars were surface sterilized with 30 % (v : v) NaClO (5.2 % available chlorine) for 30 min and then rinsed thoroughly five or six times with water. The sterilized seeds were distributed uniformly on plastic mesh grates with 1-mmAfter IA and EA treatments, roots with lengths of 5–8 cm were selected for aerenchyma measurements. Root tissue was prepared as described previously (4NO3 (LN), 1.25 mM Ca(NO3)2 (NO3-N), 1.25 mM (NH4)2SO4 (NH4-N) and 1.25 mM NH4NO3 (N/N). To inhibit nitrification, 7 µM dicyandiamide (DCD-C2H4N4) was added to the nutrient solutions. Whole roots were collected after 0.5, 2, 6, 12, 24 and 48 h of growth in the nutrient solutions to analyse gene expression. Shoots and roots of rice seedlings were, respectively, collected 48 h later to measure biomass and total N concentration. Plant samples were desiccated in a forced-air oven at 70 °C for ∼72 h to a constant weight, after which their dry weight was measured. The measurement of total N contents was performed according to Rice seedlings of identical height cultivated under IA and EA conditions for 5 days were selected for N treatments for a further 2 days. Seedlings were divided into five groups for treatment under different N conditions. The seedlings were grown in tanks containing 10 L of nutrient solution. The five N treatments were listed as no N (−N), 0.125 mM NHThe ethylene levels of five seedlings of the two rice varieties were determined after aeration and N treatments. Seedlings were transferred into 10 mL gas chromatography (GC) vials . We then added 4 mL of the same N solution as used for the N treatments into IA group vials or waterman filter paper soaked with same N solutions into EA group vials and collected ethylene released from the seedlings for 12 h. The 6 mL total air in these vials was introduced into 50 mL syringes and subjected to GC analysis  as described previously (http://www.invitrogen.com). For quantitative PCR (qPCR) analysis, total RNAs were treated with DNaseI and reverse transcribed using SuperScript II (Invitrogen) to produce cDNA. Triplicate quantitative assays were performed on each cDNA dilution using SYBR Green Master Mix and an ABI 7000 sequence detection system according to the manufacturer's protocol . Gene-specific primers were designed using the Primer Express software (Applied Biosystems). The relative quantification method was used to evaluate the quantitative variation between replicates. Amplification of OsRAc1 (Actin) was used as an internal standard to normalize all expression data. All primers used for qPCR are listed in Supporting Information—Table S1.Total RNA was prepared from the roots of YD6 and NK57 seedlings using TRIzol reagent  with aeration and time course as two main effects was used. One of the aims of the experiment was to investigate the nature of the aeration responses in rice plant. We measured adventitious root number, root surface area, aerenchyma formation, ethylene production and the expression of aerenchyma-formation- and N-related genes by aeration treatment of each cultivar individually. These data except gene expression were compared with the differences between cultivars under aeration treatment with two-way repeated-measures ANOVA. For the gene expression of each cultivar, one-way ANOVA was used to test significance of data difference. In order to study the interaction between aeration and N treatment on rice response, we used a full model factorial experiment ANOVA to test for main and interactive effects of cultivar, aeration and N treatments on rice dry weight, total N concentration and ethylene production. Furthermore, we investigated the influence of aeration during treatment time course on aerenchyma-formation- and N-usage-related gene expression in two rice varieties under LN and NO3-N conditions. The significant differences of gene expression over aeration time under different N conditions were determined by the analysis of covariance (ANCOVA).All statistical evaluations were conducted using IBM SPSS ver. 13 software. To determine whether dissolved O2 level on root growth, we quantified changes in the dissolved O2 level of the rice-growing solutions after EA. Under IA conditions, the dissolved O2 decreased from 4.3 to 3.8 mg L−1 by 48 h and stabilized at 3.6 mg L−1 after 120 h of growth . This effect was particularly noticeable in the sections obtained 1.5 cm from the root tip, in which aerenchyma formation was reduced from ∼16 to 0 % by EA in both cultivars . In the sections obtained 2.5 cm from the root tip, aerenchyma formation was decreased in NK57 roots by ∼9 % . Aerenchyma formation was significantly different between cultivars and between aeration treatments but not between cultivar and aeration treatment  were determined under −N supply. OsNRT2.1 expression was increased by ∼30 % after EA treatment in both cultivars  with different aerenchyma formation. The number of adventitious roots, root surface area and root dry weight were increased by EA in YD6 and NK57 five days after germination  and PAPD (0306J0802).This research was funded by the 2 measurement. Q.S. and G.X. were involved in editing the manuscript.J.Z. and J.L. conducted all the experiments. Z.X. was involved in ethylene measurement. X.F. designed the experiments and wrote the manuscript. Q.Z. was involved in soluble ONone declared.The following additional information is available in the online version of this article –Table S1. Primers and accession numbers used for quantitative real-time PCR.Figure S1. Effects of aeration on aerenchyma formation of 7-day-old seedlings. Transverse section of root visualized in resin-embedded sections of YD6 (A) and NK57 (B) roots. Resin-embedded sections obtained 1.5 and 2.5 cm from the root tips of YD6 and NK57 seedlings subjected to IA and EA treatments. Quantification of aerenchyma formation in sections obtained 1.5 cm (C) and 2.5 cm (D) from the root tips, using Image J software. Values represent the means ± SE (error bars) of three replicates. Significant differences are indicated by different letters .Figure S2. Real-time quantitative RT-PCR analysis of OsPAD4 and OsLSD1.1 expression in roots of rice seedlings grown in N/N and NH4-N nutrient solution. Time course of OsPAD4 expression in N/N (A) and NH4-N (B) nutrient solution. Time course of OsLSD1.1 expression in N/N (C) and NH4-N (D) nutrient solution. Seedlings grown with or without aeration were transferred to a LN nutrient solution and roots were collected for gene expression analysis at 0, 0.5, 2, 6, 12, 24 and 48 h. IA, internal aeration; EA, external aeration. Values represent the means ± SE (error bars) of three replicates.


Systemic lupus erythematosus (SLE) is a multifactorial disease. Rare causes of monogenic SLE have been described, including the complement deficiencies.Our objectives were to collect clinical data and outcome of SLE patients associated to complement deficiency in a multicenter retrospective study.We conducted a retrospective study within the French paediatric rheumatology society (SOFREMIP) in 2012-2013.to identify patients, with a confirmed deficiency of complement fraction.Ten cases of SLE with complement deficiency were identified: 2 C1 deficiencies, 2 C2 deficiencies and 6 partial C4 deficiencies. The sex ratio (M/F) is 0/10. The disease onset occurred in childhood in 8 patients with 6 before the age of 10. The first symptoms were cutaneous in 7 children, articular for 2 children and psychiatric for 1 patient. All patients were positive for antinuclear antibodies whereas only half of them were positive for anti-dsDNA antibodies. Anti-Ro (SS-A) antibodies were strongly positive in 8 patients. Anti-phospholipidantibodies were presentin 6 patients. Over time, 5 patients developed a severe disease associated to renal failure (n = 2) or neurolupus (n = 3). Associated autoimmune diseases were found in 4 patients: hypothyroidism (n = 1), autoimmune hepatitis (n = 2), Sjögren syndrome (n = 1). Two children with C2 and C4 deficiency had severe bacterial infections.Cutaneous or joint manifestations are the most common symptoms but life-threatening complications can occur in the context of C1 deficiency. Anti-SSA antibodies were frequent while anti-DNA are only found in half of the cases. Genetic characterization of complement deficiencies remains challenging. Next generation sequencing may be helpful to better diagnose these monogenic forms of lupus.None declared.


The emergence of severe cases of human influenza A (H7N9) viral infection in China in the spring of 2003 resulted in a global effort to rapidly develop an effective candidate vaccine. In this study, a cold-adapted (ca), live attenuated monovalent reassortant influenza H7N9 virus (Ah01/AA ca) was generated using reverse genetics that contained hemagglutinin (HA) and neuraminidase (NA) genes from a 2013 pandemic A H7N9 isolate, A/Anhui/01/2013 virus (Ah01/H7N9); the remaining six backbone genes derived from the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AA virus). Ah01/AA ca virus exhibited temperature sensitivity (ts), ca, and attenuation (att) phenotypes. Intranasal immunization of female BALB/c mice with Ah01/AA ca twice at a 2-week interval induced robust humoral, mucosal, and cell-mediated immune responses in a dose-dependent manner. Furthermore, the candidate Ah01/AA ca virus was immunogenic and offered partial or complete protection of mice against a lethal challenge by the live 2013 influenza A H7N9 (A/Anhui/01/2013). Protection was demonstrated by the inhibition of viral replication and the attenuation of histopathological changes in the challenged mouse lung. Taken together, these data support the further evaluation of this Ah01/AA ca candidate vaccine in primates. Since the first notification at the end of March 2013, China has been reporting cases of human infection with H7N9 virus to the World Health Organization. This outbreak was the first identification of infection with this virus in humans [http://www.who.int/influenza/vaccines/use/en/). Unlike the inactivated influenza vaccine, a live attenuated influenza vaccine (LAIV) is administered intranasally (i.n.), and more closely mimics the natural process of influenza virus infection. Previous researches have revealed that LAIV induces robust humoral, mucosal and cell-mediated immune responses, thus providing strong protective efficacy and long-lasting immunity [Vaccination is the most effective method of preventing infection by influenza viruses and the severe outcomes thereof. Safe and effective vaccines have been available and used for more than 60 years . Among himmunity . In addiimmunity .ca, ts and att reassortant influenza A H1N1 viruses in a short period of time using reverse genetic technology. The cold-adapted, attenuated, and live 2009 pandemic H1N1 vaccines elicit a high level of antibodies response that provide effective protection against wt H1N1 virus infection in animal models, demonstrating the potential of CAIVs [To date, three cold-adapted influenza A virus strains, A/Ann Arbor/6/60 (AA) (H2N2), A/Leningrad/134/17/57 (H2N2), and A/Leningrad/134/47/57 (H2N2), and two B virus strains, B/Ann Arbor/1/66 ca and B/USSR/60/69 ca have been developed as a LAIV Master Donor virus to generate seasonal influenza vaccines for clinical use in humans [in vitro and in vivo. Thus, our preclinical data support the further evaluation of the Ah01/AA ca vaccine in clinical trials.In this study, a live attenuated A H7N9 virus was generated containing the HA and NA genes of A H7N9 virus (A/Anhui/01/2013) isolated at the beginning of the 2013 influenza A H7N9 virus outbreak in China in the background of the A/Ann Arbor/6/60 ca virus using reverse genetics. Phenotypes and protective efficacy of the candidate Ah01/AA ca virus was evaluated Female BALB/c mice, 4–6 weeks old, were purchased from the Laboratory Animal Center of Beijing Institute of Microbiology and Epidemiology and housed under standard temperature, light, and dark cycles. This study was specifically approved by the Institutional Animal Care and Use Committee (IACUC) and ethics committee of Beijing Institute of Microbiology and Epidemiology (ID: SYXK2012-005). All facilities were also accredited by the Animal Care and Ethics Committee of Beijing Institute of Microbiology and Epidemiology. Live-virus experiments were performed in Bio-safety Level 3 facilities in accordance with governmental and institutional guidelines.Subject provided written informed consent for participation in this study.50) for each virus was determined by serial titration of the virus in Madin-Darby canine kidney (MDCK) cells.The human influenza A H7N9 virus  used in this study was isolated from a confirmed H7N9-infected patient in the Anhui province in 2013 . The genwt Ah01/H7N9. Detailed protocols were performed as previously described [For each A H7N9 reassortant virus, the six plasmid DNAs encoding the internal protein genes of the AA ca were combined with the two plasmids encoding the HA and NA surface antigen genes from escribed . The allca and ts phenotypes of the Ah01/AA ca virus were determined as described in detail previously [The eviously , 12.6 CCID50 of either the Ah01/H7N9 or Ah01/AA ca virus. Three days p.i., nasal turbinates, lungs, and brains of infected mice were collected and homogenized in DMEM medium to produce a 10% w/v tissue homogenate. Next, tissue homogenates were obtained by centrifugation and were titrated in MDCK cells. Viral titers of tissue were calculated and expressed as Log CCID50/g.BALB/c mice (n = 5) under sodium pentobarbital anesthesia (60–80 mg/kg) were inoculated i.n. with serial 10-fold dilutions of the Ah01/AA ca virus to determine the pathogenicity. The survival rates of mice were monitored and recorded daily until 14 days. Mice showing 30% of body weight loss were considered to have reached the experimental end point and were humanely euthanized. To evaluate viral replication in various organs, mice were inoculated i.n. with 104 CCID50, 105 CCID50, or 106 CCID50 of each reassortant virus in a total volume of 20 μl, respectively, and sera were collected at 2 weeks after prime and boost. The serum hemagglutination-inhibition (HI) titers were determined by standard methods using 4HA units’ viruses. Serum neutralization antibody titers were tested by micro-neutralization (MN) assays as described previously [Groups of mice under sodium pentobarbital anesthesia (60–80 mg/kg) was immunized i.n. twice, in 14 days apart, with different viral doses containing 10eviously . Nasal w4 CCID50, 105 CCID50, or 106 CCID50 of Ah01/AA ca virus or PBS (mock-immunized) in a total volume of 20 μl in two doses spaced 2 weeks apart. Mice under sodium pentobarbital anesthesia (60–80 mg/kg) were challenged with the wt Ah01/H7N9 virus (50-fold the LD50) 14 days after the second immunization. Challenged mice were monitored and recorded daily in terms of body weight and survival for 14 days. In this experiment, no animals exhibit any adverse health or behavior symptoms without intervention. Notably, 10% animals in control group that died in the survival study reached the humane endpoint. Mice showing > 30% of body weight loss was considered to have reached the experimental end point were humanely euthanized by cervical dislocation and then carefully observed and palpated to ensure that there was no respiration and heartbeat. Lung tissues were collected, homogenized, and titered using MDCK cells at 3 days post-challenge. Viral titers were calculated using the Reed and Muench method and are expressed as log10 CCID50/g lung tissue. Histopathological changes in the lung were assessed at 3dpi by H&E staining and subsequent visualization under a microscope.Mice were immunized i.n. with 10The levels of mucosal IgA antibodies against influenza A (H7N9) antigen were determined separately by indirect enzyme-linked immunosorbent assays. Notably, 96-well ELISA plates were coated with 400 ng of purified H7N9 viral antigens. Detailed protocols were performed as previously described . This exAll statistical analyses were performed using the GraphPad Prism software. All data are expressed as means ± SD unless otherwise stated. Following an ANOVA, a multiple comparison test was also performed using the GraphPad Prism ver. 5.0 software.10 CCID50/ml for the Ah01/AA ca. The reassortant Ah01/AA ca viral particles were examined by electron microscopy and exhibited spherical structures 80–120 nm in size and a typical lipid membrane bilayer on their outer surface  A/Ann Arbor/6/60 (H2N2) virus exhibit the enotypes –16. The  at 25°C . These dwt Ah01/H7N9 virus in both the nasal turbinates and lungs of infected mice were highly elevated . In contrast, Ah01/AA ca replication was restricted to the upper respiratory system . Furthermore, no reassortant Ah01/AA ca virus replication was detected in spleen, kidney, and brain at 3 days post inoculation. In addition, inoculation of mice with the reassortant viruses did not result in death, even at the highest dose used (data not shown here).As shown in 6 CCID50 group. A significant difference in HI titers was observed between the Ah01/AA ca vaccinated group and the PBS control group. Additionally, 14 days following the second vaccination, the HI antibody titer increased sharply to 1,280 in the 106 CCID50 group. The serum neutralizing antibody levels were also measured in the mice. Serum NT titers against wt Ah01/H7N9 virus were 60, 180, and 280 in the 104 CCID50, 105 CCID50, and 106 CCID50 vaccine groups, respectively, following two doses of vaccine. Concomitantly, the increase in NT titers in mouse sera corresponds with the change in HI titers.Humoral immune responses induced by i.n. immunization of Ah01/AA ca vaccines were assessed by assaying HI and NT antibodies in the serum of vaccinated mice. As shown in 6 CCID50 Ah01/AA ca elicited a stronger sIgA antibody response in the lung lavage fluid compared to that in the nasal lavage fluid in response to the Ah01/H7N9 virus   . These din vitro. As shown in p<0.001). Of particular note, no significant difference in the number of cytokine-producing T cells was observed between the 106 CCID50 and 105 CCID50 vaccinated groups. Additionally, treatment with the Ah01/AA ca vaccine induced significantly more IL-4–producing T cells, in a dose-dependent manner (p<0.001), compared to the control group  at 2 weeks after boost. In the 106 CCID50 vaccinated group 100% mice survived for at least 14 days following challenge with the Ah01/H7N9 influenza virus, and their body weight recovered rapidly at 6 days post infection  virus. Influenza H7N9 viruses can infect humans without prior adaptation, suggesting that they likely present a potential pandemic threat [


In particular, they failed to form cystic embryoid bodies and showed defects in primitive endoderm formation. To pinpoint the underlying mechanism, we undertook a structure-function approach. Rescue of p120ctn-null mESCs with different p120ctn wild-type and mutant expression constructs revealed that the long N-terminal domain of p120ctn and its regulatory domain for RhoA were dispensable, whereas its armadillo domain and interaction with E-cadherin were crucial for primitive endoderm formation. We conclude that p120ctn is not only an adaptor and regulator of E-cadherin, but is also indispensable for proper lineage commitment.E-cadherin-mediated cell-cell adhesion is critical for naive pluripotency of cultured mouse embryonic stem cells (mESCs). E-cadherin-depleted mESC fail to downregulate their pluripotency program and are unable to initiate lineage commitment. To further explore the roles of cell adhesion molecules during mESC differentiation, we focused on p120 catenin (p120ctn). Although one key function of p120ctn is to stabilize and regulate cadherin-mediated cell-cell adhesion, it has many additional functions, including regulation of transcription and Rho GTPase activity. Here, we investigated the role of mouse p120ctn in early embryogenesis, mESC pluripotency and early fate determination. In contrast to the E-cadherin-null phenotype, p120ctn-null mESCs remained pluripotent, but their  Disease may be due to either excess of undesirable cells, like in cancer or autoimmune disease, or by progressive loss of vital cells. The latter, for instance, causes neurodegenerative diseases such as Alzheimer’s disease. Stem-cell based therapy holds great potential to cure devastating diseases with cell loss or dysfunctionality, because stem cells have the capacity to form any given cell type of the body. Recent advances in the field allow to obtain stem cells from virtually every patient. These stem cells could then be instructed to form the desired cells that can be reintroduced to cure the patient. Before such therapies are suited for the clinic, we first need comprehensive knowledge of the molecular mechanisms that underlie cell fate decisions. Here, we scrutinize the role of a junctional protein, called p120ctn, in both stem cells and lineage-committed cells. Importantly, this key protein has a modular structure, and each of its segments has different interaction partners and biological functions. We deleted p120ctn specifically in stem cells, and reintroduced several p120ctn mutants that lack specific protein segments. As such, we could unravel the exact molecular interaction that is required for p120ctn to drive the differentiation of stem cells towards primitive endoderm. Pluripotent mouse embryonic stem cells (mESCs) can self-renew and differentiate into any given cell type within an organism. They are isolated from the inner cell mass (ICM) of preimplantation blastocyst stage embryos and considered ‘naive’, with regard to their pluripotency status, whereas stem cells derived from the epiblast of the post-implantation embryo are considered ‘primed’ . Naive min vitro by allowing pluripotent mESCs to differentiate into three-dimensional cell aggregates grown in suspension, known as embryoid bodies (EBs) . Th. Th-/-;Aeted EBs . We usedeted EBs .in vitro structure-function studies in a p120ctn-null background.The rescue constructs included a series of wild-type p120ctn isoforms, p120ctn mutants or downstream effectors. In total, we report here on 19 different rescue constructs, used for generation of 133 targeted mESC clones . This ocMultiple p120ctn isoforms  Fig 4D)Fig 4D) wAs the results of the previous experiments suggest that the armadillo domain and the C-terminal region of p120ctn are sufficient for proper mESC differentiation, we studied the binding motifs for E-cadherin and RhoA, which are embedded in the armadillo domain. Two RhoA binding domains in p120ctn  or dominant-negative (DN) versions of each of the representative Rho GTPase family members, RhoA, Rac1 and Cdc42. In all these rescue attempts, the mutant mESCs failed to differentiate into cystic EBs  for 4 days. For each mESC line, 12 puromycin-resistant colonies were picked, expanded and screened by PCR 1 Jae strain) treated with mitomycin C . mESC isolation from blastocysts and subsequent culture was performed according to Pieters et al. . mESCs wd by PCR , and find by PCR .EcoRI and OliI digestion from various eukaryotic expression vectors [EcoRI and EcoRV. hp120ctn Δ622–628 mutants were created with a QuikChange II Site-Directed Mutagenesis Kit (Stratagene). p120ctn K401M mutants and p120ctn K401M-CAAX mutants were generated by cloning synthetic p120ctn DNA fragments (Genescript) into pENTR1A-hp120ctn 1A and 3A vectors. The pENTR3C-hEcadh-withSTOP plasmid with a native stop codon was obtained by using QuikChange (Agilent Technologies) on pENTR3C-hEcadh-NoSTOP (kind gift from Philippe De Groote). pENTR vectors containing constitutively active or dominant-negative Rho GTPases were a kind gift from Prof. Christophe Stove (Ghent University).pENTR1A-hp120ctn 1A, 1AC, 1AΔ622–628, 3A, 3AC, 1AΔ622–628, 4A and 4AΔ622–628 were generated by ligation of p120ctn cDNA fragments, obtained by  vectors , into pE-/-;ALtg/+) mESCs were generated by crossing p120ctn+/- mice with ALtg/+ mice [-/-;ALtg/+ mESCs were cotransfected with the pRMCE-DV1 vector, from which the floxed stop cassette was removed by Cre [RMCE-compatible p120ctn-null -/-;ALtg/d by Cre , and a Fd by Cre  in a 1:1d by Cre . Table 16 cells/well, followed by culture for 4 days in SR-mESC medium and staining for alkaline phosphatase (AP) using the Leukocyte Alkaline Phosphatase Kit (Sigma).The procedure for generating EBs was similar to that described before . For marThe normal mouse mitotic karyotype consists of 40 acrocentric chromosomes. This was checked in our mESC lines by routine procedures . Chromos6 cells were injected subcutaneously into the flank of immunocompromised nude mice. Growing teratomas were isolated 5 to 8 weeks later and processed for histological analysis.Control and p120ctn-null mESCs were trypsinized and 1.5 x 102. The next day, blastocysts were transferred into the uteri of 2.5-dpc pseudopregnant Swiss females previously mated with vasectomized males. Chimeras were identified at birth by the presence of black eyes and later by agouti coat pigmentation.Chimeras were generated by diploid embryo aggregation. Briefly, E2.5 embryos were collected from superovulating Swiss female mice and the zona pellucida of embryos was removed by treatment with acid Tyrode's solution (Sigma). mESC colonies from control and p120ctn-null mESCs were briefly treated with 0.25% trypsin-EDTA (Invitrogen) to form loosely connected clumps of 7 to 10 cells. Each zona-pellucida free embryo was aggregated with a clump of 7 to 10 cells using depression wells made with an aggregation needle  in a 35-mm plastic dish . Aggregates were cultured overnight in microdrops of KSOM with amino acids (Biognost) under mineral oil (Sigma) at 37°C in 95% air and 5% CO4 with 1.5% K3Fe(CN)6 in 0.1 M sodium cacodylate buffer at room temperature for 1 hour. After washing in double distilled H2O, cells were subsequently dehydrated through a graded ethanol series, including a bulk staining with 1% uranyl acetate at the 50% ethanol step followed by embedding in Spurr’s resin. Ultrathin sections of a gold interference color were cut using an ultra-microtome (Leica EM UC6), followed by a post-staining in a Leica EM AC20 for 40 min in uranyl acetate at 20°C and for 10 min in lead stain at 20°C. Sections were collected on Formvar-coated copper slot grids and were viewed with a JEM 1010 transmission electron microscope  operating at 60 kV using Image Plate Technology from Ditabis .EBs were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 for 4 hours at room temperature followed by fixation overnight at 4°C. After washing in buffer, they were post fixed in 1% OsO2O2 in methanol. Sections were then treated with 1% goat serum/1% BSA in PBS, followed by incubation with primary antibodies  or overnight (for mESCs) with primary antibody , followetibodies  overnighHmbs, Actb and Gapdh. All primers are listed in Total RNA was isolated using RNeasy Plus Mini Kit (QIAGEN). cDNA was synthesized using the First Strand cDNA Synthesis Kit (Roche) with oligo(dT) primer starting from equal amounts of RNA as measured by a NanoDrop spectrophotometer (Thermo Scientific). qRT-PCR was performed using the LightCycler 480 SYBR Green I Master (Roche) and monitored on a LichtCycler 480 system (Roche). Gene expression was standardized against reference genes SacII site and a 3ʹ XbaI site (underlined) flanking the NLS sequence, which is interrupted by p120ctn exon C : 5’- CCGCGGAAGAAGGGCAAAGATGAGTGGTTCTCCAGAGGGAAAAAGCCTTCTAGA-3’; 3’-GGCGCCTTCTTCCCGTTTCTACTCACCAAGAGGTCTCCCTTTTTCGGAAGATCT-5’. The oligonucleotides were annealed, digested with SacII and XbaI, and ligated in pHM829 pre-digested with the same enzymes.The mammalian expression vector pHM829 was designed for expression of proteins of interest fused to β-galactosidase  at its N-terminus and to green fluorescent protein (GFP) at its C-terminus . pHM829 2 in DMEM supplemented with 10% fetal bovine serum, 4 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 mg/ml). Cells were transfected by using Fugene reagent (Roche Applied Science). For each construct tested in the branching assay, at least 100 transiently transfected HeLa cells were scored for normal or branched cellular phenotype.HeLa cells were grown at 37°C under 5% COAn antibody specific for p120ctn isoform C (pAb exC) was generated in rabbits against a peptide containing the six AA encoded by alternative exon C (GKDEWFSRGKGAC) and fused to keyhole limpet hemocyanin . This serum was characterized and validated by immunofluorescence of cells transiently transfected with either pEFBOS hp120ctn 3A or 3AC , and by mESCs were lysed in buffer containing 150 mM NaCl, 2 mM EDTA, 25 mM Tris-HCl, pH 8 and 0.5% Triton-X-100 supplemented with Protease Inhibitor Cocktail (Roche). The insoluble fraction was removed by centrifugation and the Bio-Rad DC assay kit was used for protein measurement of the lysate. 800 μg of protein was incubated for 2 h at 4°C with 5 μg of antibody, either mouse anti-p120ctn (BD Transduction) or, as an irrelevant antibody control, mouse anti-GFP (Invitrogen). After antibody incubation, 50 μl of protein-G Dynabeads (Invitrogen), previously washed in lysis buffer, were added and incubation was continued for 1 h at 4°C. The bead–protein complexes were washed 4 times with lysis buffer and a magnetic rack was used to collect the beads. Proteins were eluted from the beads by boiling the samples in SDS-loading buffer.S1 Fig(A) Confocal fluorescent images of a wild-type blastocyst stained for p120ctn and the apical marker PKCζ. p120ctn is expressed basolaterally. Scale bar: 25 μm. (B) Maternal p120ctn allows basal E-cadherin stabilization on the membranes of p120ctn-deficient blastocysts. Three-dimensional (3D) reconstruction using consecutive confocal sections of the blastocysts shown in -/-, arrowheads) probably allows the stabilization of basal E-cadherin levels (arrowheads) on the membranes of blastocysts. This is thought to be sufficient for normal compaction and blastocyst formation. Scale bar: 25 μm.(PDF)Click here for additional data file.S2 Fig(A) Chromosomal analysis of control and p120ctn-null mESCs. Fluorescent images of DAPI-stained mitotic spreads with 40 acrocentric chromosomes from control mESC lines , p120ctn+/+ (+/+9), p120ctn+/- (+/-1)) and p120ctn-null mESCs . (B) Graph depicts the percentage of mitotic spreads that contain 39, 40, 41, 42 and 43 or more chromosomes. An mESC line is considered normal if 70% or more of its spreads contain 40 chromosomes. The names of mESC lines analyzed are at the bottom. (C) Confocal fluorescent images of control and p120ctn-null mESCs stained for α-catenin and β-catenin. The boxed areas were further magnified 3.6-fold. Scale bars: 25 μm.  qRT-PCR analysis for expression of various stemness genes, in (D) control and p120ctn-null mESCs, and in (E) their corresponding EBs after 30 days of culture (DIV30). Actb, Gapdh and Hmbs were used as reference genes. The error bars in the graphs represent the standard error of the mean of two independent control or p120ctn-null cell lines.(PDF)Click here for additional data file.S3 Fig+/+) and p120ctn-depleted (p120ctn-/-) mESCs. Scale bars: 100 μm.p120ctn loss does not abrogate germ layer development. Histological analysis of H&E stained sections of teratomas from control (p120ctn(PDF)Click here for additional data file.S4 Fig and p120ctn-null EBs (C-E). The red dashed box in (D) is enlarged in (E) and shows a region of minimal endodermal cell-cell adhesion (blue arrows) showing non-polarized microvilli (black arrows). Scale bars: 2 μm. AJ, adherens junction; DS, desmosome; TJ, tight junction.TEM analysis of DIV12 control EBs (PDF)Click here for additional data file.S5 Fig(A) Scheme depicting our mouse breeding protocol, followed by isolation of the RMCE-compatible p120ctn-/-;ALtg/+ mESCs, and insertion of various rescue cDNAs in the ROSA26 locus by RMCE. By Gateway cloning we inserted a set of candidate rescue cDNAs  into the ROSA26 locus and in addition restored neomycin-resistance. In these targeted mESCs, both the GOI and NeoR genes are driven by the endogenous R26 promoter. A fluorescent image of a DAPI-stained mitotic spread with 40 acrocentric chromosomes from p120ctn-/-;ALtg/+ mESCs is shown at the bottom. (B) Graph depicting p120ctn levels in control and p120ctn-null mESC, and in p120ctn-null mESC with R26-driven expression of p120ctn isoform 1A (R_p120_1A) or of its K401M mutant (R_1A_K401M). Z-stacks, optimized according to the Nyquist sampling theorem, were acquired on the SP5 Leica confocal microscope. A fixed intensity threshold was set on the Alexa 488 signal representing p120ctn staining. Within this threshold, the total amount of voxels for each mESC colony was counted and normalized against its total nuclear volume. At least 10 reconstructed colonies were analyzed for each mESC line. (C) Confocal fluorescent images of p120ctn-null mESCs with R26-driven expression of p120ctn isoform 3A (R_p120_3A) or 4A (R_p120_4A) stained for p120ctn or E-cadherin expression. A threefold magnified image is shown below each picture. Scale bars: 50 μm.(PDF)Click here for additional data file.S6 Fig(A) Nuclear translocation assay using fusion proteins composed of an N-terminal β-galactosidase  part and a C-terminal GFP. Between the β-gal and the GFP parts we cloned the NLS of p120ctn , a mutated version of it (NLSmut), or the NLS interrupted by amino acids encoded by exon-C (NLSexon-C). These constructs were expressed in HeLa cells. Confocal fluorescence analysis showed that both NLSmut and NLSexon-C prevented the nuclear GFP expression seen with the NLS construct. The exon-C encoded AA expressed by the NLSexon-C construct were also specifically detected by an in-house made polyclonal antibody .  Branching assay in HeLa cells transiently transfected with plasmids expressing either (B) p120ctn isoform 1A and related proteins, or (C) isoform 3A and related proteins. The related proteins were p120ctn isoform C variants (p120ctn isoforms 1AC and 3AC), or mutants lacking amino acids 622–628 (p120ctn isoforms 1AΔ and 3AΔ). Confocal images were made after immunostaining for all p120ctn isoforms , either combined or not combined with specific staining for p120ctn isoform C . (D) Graph showing the percentage of branched versus normal cellular phenotypes upon overexpression of different p120ctn isoforms in HeLa cells, as illustrated in panels (B) and (C). (E) Micrographs of DIV30 p120ctn-null EBs with R26-driven expression of p120ctn isoform 1AC (R_p120_1AC) or 3AC (R_p120_3AC). Two independently rescued mESC lines were analyzed in each setup. Scale bars: 200 μm. (F) Confocal fluorescent pictures of control and p120ctn-null mESCs stained for p120ctn (green) and RhoA (red). A 3.3-fold magnification is shown below each picture. Scale bars: 15 μm. (G) Micrographs of DIV30 p120ctn-null EBs with R26-driven expression of dominant-negative (DN) or constitutively active (CA) Rho GTPases, as indicated. Two independently rescued mESC lines were analyzed in each setup. Scale bars: 200 μm.Amino acids encoded by p120ctn exon-C inhibit nuclear translocation and dendritic-like branching. (PDF)Click here for additional data file.S7 Fig(A) Confocal fluorescent pictures of R_p120_3A mESCs, and of mESCs expressing the K401M mutant of p120ctn isoform 3, after immunostaining for p120ctn and E-cadherin. Scale bars: 25 μm. (B) Micrographs depicting the morphology of different mESC cultures, as indicated. Scale bars: 100 μm(PDF)Click here for additional data file.S8 Fig(A) Immunostaining to characterize an antibody specific for isoform C of p120ctn (pAb ExC). This antibody recognizes in transiently transfected MCF7 cells human p120ctn isoform 3AC, but not human isoform 3A lacking exon-C encoded amino acids. Monoclonal antibody pp120 recognizes all p120 isoforms. (B) Western blot analysis of two wild-type gastrulating mouse embryos (E7.5) in which all endogenous p120ctn isoforms were detected with pp120 antibody and endogenous p120ctn isoforms C were detected with pAb ExC. Mouse brain was taken as a positive control because it is relatively rich in isoforms C of p120ctn.(PDF)Click here for additional data file.


Various strategies involving the use of hepatitis C virus (HCV) E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system. Hepatitis C virus (HCV) infection, which affects an estimated 170 million people worldwide and frequently leads to severe chronic liver disease, constitutes a major global health concern , but theThe immunological correlates of the successful control of HCV infection and the underlying mechanism remain unclear, but many studies in both humans and chimpanzees have demonstrated that the early establishment of vigorous, broadly cross-reactive, long-lasting CD4+ and CD8+ T-cell responses is associated with spontaneous HCV clearance , 6. It iWe recently proposed an original vaccination strategy based on HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus (HBV) S envelope protein and self-assembling into highly immunogenic, noninfectious and secreted subviral envelope particles resembling the HBV vaccine , 25. TheIn this study, we used our chimeric HBV-HCV envelope particle model to compare the use of E1 and E2 as separate immunogens with their use in the form of the E1E2 heterodimer, in terms of immunogenicity and the capacity to induce neutralizing antibodies. We found that anti-E1 and anti-E2 antibodies had additive neutralizing properties, and that the best strategy for inducing a dual anti-E1 and anti-E2 response is to immunize with a mixture of vaccine particles bearing E1 and E2 separately.All animal immunizations were conducted by the company Agro-Bio , accredited by the French Administration to conduct protocols on laboratory animals in accordance with European Directive 2010/63/EU. The protocol was approved by the Agro Bio Ethic Committee n° C2A-99 .Chimeric HBV (adw genotype)-HCV (1a genotype) envelope subviral particles bearing the entire HCV E1 and/or E2 proteins  were produced and purified, as previously described , 25. FolAnti-HBsAg antibodies were quantified with the ARCHITECT system (Abbott Laboratories). Anti-E1 and anti-E2 responses were evaluated with “in-house” ELISAs, based on the use of solid phases consisting of lysates of baby hamster kidney (BHK-21) cells expressing HCV E1 or E2 proteins, which were compared with control cells expressing ß-galactosidase . For these experiments, the DNA sequences encoding HCV E1 or E2 envelope glycoproteins derived from a genotype 1a HCV isolate (isolate Dj) were therefore cloned into an original eukaryotic DNA expression vector (pSFV) based on the Semliki Forest Virus replicase complex, which present a high level of expression efficiency. These DNA sequences, amplified from the previously described pSFV-HCVdj plasmid (Genbank AF529293) encoding the core-E1-E2 sequence of a genotype 1a HCV , also coHCV in cell culture (HCVcc) harboring HCV envelope glycoproteins derived from genotype 1a (JFH1/H77), 1b (JFH1/J4), 2a (JFH1 WT), 3 (JFH1/S52) and 4 (ED43/JFH1) isolates –34, werep value < 0.05 was considered to be statistically significant.Statistical analyses were performed with nonparametric Mann-Whitney tests to evaluate the observed subtle differences in the immunogenetic potential and the capacity to induce neutralizing antibodies between immunization with a mixture of chimeric particles harboring E1 or E2 proteins separately and other vaccination strategies. A We first evaluated, with biological material collected during a previous study , the sepOur ultimate goal is to induce the best neutralizing response. We therefore investigated whether the anti-E1 and anti-E2 antibodies induced by immunization with our chimeric particles could have additive properties to increase the cross-neutralization of heterologous strains of various HCV genotypes. We compared the neutralizing properties of mixtures of equal quantities of an anti-E1 rabbit serum and an anti-E2 rabbit serum with those of these same individual sera against HCVcc harboring HCV envelope glycoproteins derived from genotype 1a, 1b, 2a and 3 isolates. In all cases, these mixed sera presented better cross-neutralizing properties in vitro against HCVcc harboring heterologous HCV envelope proteins derived from strains of different genotypes than did the individual sera . This crBased on these findings for biological material collected during our previous animal immunizations, we conducted new immunization protocols with equal-amount mixtures or various sequential combinations of AddaVax-adjuvanted chimeric particles harboring E1 and E2 proteins separately, to try to identify the vaccination protocol inducing the best dual anti-E1 and anti-E2 response. Unfortunately, one of the rabbits immunized with particles bearing the E1E2 heterodimer died during blood sampling on day 14, but this death was clearly not related to the quality of the immunogen administered. All rabbits immunized by four injections of a mixture of chimeric particles bearing E1 and E2 separately were shown to develop sustained responses against both the E1 and E2 envelope proteins of HCV, whereas rabbits immunized with different sequential combinations  essentially developed a response against the first immunogen . The mixp < 0.01), and significant, for genotypes 1a and 1b (p < 0.05). Moreover, additional neutralizing assays using five-fold serial dilutions of rabbit sera collected on days 0 and 56 demonstrated that a significant specific neutralizing activity against HCVcc could still be observed for high serum dilutions , and used as a mixture to induce an optimal dual anti-E1 and anti-E2 response neutralizing heterologous strains of various HCV genotypes. Further studies, including immunization in humans, will be required before definitive conclusions can be drawn. However, these vaccine particles, which could be produced by industrial procedures adapted from those established for the HBV vaccine, appear promising for use in an affordable potential prophylactic vaccine against HCV and, potentially, a bivalent HBV-HCV vaccine.S1 Fig(A), Western blots probed with polyclonal anti-HBsAg (R247) and monoclonal anti-E1 (A4) and anti-E2 (H52) antibodies demonstrated the efficient incorporation of chimeric HBV-HCV envelope proteins (E1-S or/and E2-S) into the secreted S+E1-S, S+E2-S and S+E1-S+E2-S subviral particles used for the preparation of the final immunogen solutions. (B), An immunoassay using human monoclonal antibodies recognizing conformation-dependent discontinuous epitopes on the E2 protein (AR3A) and the folded E1-E2 complex (AR5A) demonstrated the productive folding and heterodimerization of HCV envelope proteins in the context of fusion proteins. The monoclonal antibodies AR3A and AR5A were added to ELISA wells coated with chimeric particles  or JFH-1 WT viruses purified on sucrose cushions  captured with a lectin precoating. Specific binding was detected with monoclonal peroxidase-conjugated mouse anti-human immunoglobulin antibody. Absorbance at 490 nm was determined. The data shown are the means ± standard deviation of one experiment performed in quadruplicate.(EPS)Click here for additional data file.S2 FigGroups of six rabbits were immunized subcutaneously four times with AddaVax-adjuvanted chimeric S+E1-S+E2-S envelope particles (vaccine doses consisting of 15 micrograms of the S+E1-S+E2-S immunogen), or with a mixture  of the S+E1-S and S+E2-S subviral particles produced separately) or different sequential immunization combinations of AddaVax-adjuvanted chimeric particles harboring E1 or E2 proteins separately (vaccine doses consisting of 15 micrograms of the S+E1-S or S+E2-S immunogens), on days 0, 14, 28 and 42. For sequential immunization combinations, rabbits were either immunized twice with S+E1-S particles and then twice with S+E2-S particles, or vice versa. Groups of three rabbits immunized with the Addavax adjuvant alone or with Engerix (vaccine doses consisting of 15 micrograms of the S immunogen), a commercially available HBV vaccine, were used as negative and positive controls, respectively. Serum samples were collected from rabbits at various time points , to characterize the antibody responses.(EPS)Click here for additional data file.S3 FigDilutions of rabbit sera collected on days 0 and 56 were first incubated with HCVcc harboring HCV envelope glycoproteins derived from strains of various genotypes for 1 hour at 37°C, which were then allowed to infect Huh7.5 cells for 6 hours. After 48 hours of incubation at 37°C, the percentage neutralization was determined by subtracting the infectivity titer obtained with pre-immune serum (day 0) from that obtained with post-immune serum (day 56). The assay was performed in duplicate, and the results are expressed as mean values.(EPS)Click here for additional data file.


Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens. Respiratory syncytial virus (RSV) causes significant morbidity and mortality in children, yet an efficacious vaccine remains unavailable. Antibodies that preferentially recognize the prefusion conformation of the fusion (F) glycoprotein, particularly those that bind antigenic site Ø at the membrane-distal apex, potently neutralize infection and have aided vaccine design. Here we characterize AM14, a potent human antibody, which we show recognizes a novel epitope midway between the membrane-proximal region and the apex of the prefusion F trimer. The epitope is evenly distributed across two protomers, causing AM14 to be uniquely trimer-specific and, surprisingly, cleavage-dependent. These results indicate that the prefusion trimer is antigenically distinct from the monomer. Our findings also demonstrate that epitopes other than site Ø can be the target of extremely potent neutralizing antibodies and thus provide a new target for structure-based vaccine design. Recognition of this novel epitope could make AM14 an ideal candidate for strategies that combine passive prophylaxis with vaccination, since binding of AM14 would not block elicitation of antibodies against site Ø. Due to its unique specificity, AM14 will also be valuable for probing the conformation of RSV F-based vaccine antigens designed to be in the furin-cleaved trimeric prefusion conformation. Respiratory syncytial virus (RSV) is a ubiquitous paramyxovirus that infects nearly all children in the U.S. by two years of age . In infa2 and F1, respectively, are connected by two disulfide bonds to form a single protomer [Most RSV vaccine candidates contain at least one of the two viral surface glycoproteins: the fusion protein (F) and the attachment protein (G). Of these, only F is absolutely required for infection , and it protomer –18. Alth1 N-terminus assemble into a long alpha helix that extends toward the target cell. This reorganization pulls the hydrophobic fusion peptide from the interior of the trimer and thrusts it into the cellular membrane, resulting in a prehairpin intermediate. The F1 C-terminus then migrates toward the N-terminus, irreversibly forming a six-helix bundle and driving fusion of the two membranes [To facilitate virus entry, the mature F protein is triggered to undergo a transition from a metastable prefusion conformation to a stable postfusion conformation, resulting in fusion of the viral and host-cell membranes. Crystal structures of both prefusion and postfusion F have recently been solved, providing molecular insight into this dramatic structural rearrangement –19. Uponembranes . This reembranes .RSV-neutralizing antibodies that bind both the pre- and postfusion conformations of F were the first to be isolated. These include antibodies 101F and the murine precursor of palivizumab, antibody 1129 , 22. MorAlthough site Ø antibodies were originally thought to recognize quaternary epitopes on the prefusion trimer, they have subsequently been shown to react with monomers . Here we50s of 13.6 ng/ml for strain A Long, 12.4 ng/ml for strain A2, 30.8 ng/ml for subtype B strain 18537 and 4.6 ng/ml for subtype B strain 9320  [Microneutralization assays were performed to test the ability of AM14 to neutralize infection of HEp-2 cells by various RSV strains. AM14 potently neutralized all RSV strains tested, with ICain 9320 . For comectively . Clinicaectively . AM14 nevizumab) , 28. Simvizumab) , suggest50s of 0.63 nM and 0.18 nM, respectively  or postfusion (F ΔFP) conformation , 26. In ectively . In contectively .KD) of 0.18 nM, with rapid association and dissociation rate constants of 1.87 x 107 M-1s-1 and 3.4 x 10-3 s-1, respectively. This is in contrast to D25, which bound prefusion RSV F with association and dissociation rate constants more than 10- and 30-fold slower, respectively (1.35 x 106 M-1s-1 and 9.65 x 10-5 s-1)  . TherefoP212121 diffracted X-rays to 2.0 Å, and after a molecular replacement solution was obtained, the structure was built and refined to an Rwork/Rfree of 18.6%/22.6% . After three rounds of selection in HEp-2 cells, four unique AM14-escape viruses were isolated and sequenced. Three of the viruses each contained a single mutation in RSV F , whereas the fourth virus contained three mutations in RSV F (I79M/R429S/H515N) . ResistaMapping of the MARMs on the prefusion F structure revealed that L160S and N183K are located on the loops connecting α2 with α3 and β3 with β4, respectively, in agreement with the crystal structure . This veThe crystal structure and MARMs suggested that AM14 is a quaternary-specific antibody with an epitope spanning two protomers. To test the quaternary specificity, we measured binding of immobilized AM14 to monomeric F and trimeric prefusion F by ELISA . The mon2 subunit and the neighboring protomer, as well as differences in peptide structure compared to the complete prefusion F. Collectively, these results demonstrate that AM14 is unique in its ability to discriminate cleaved, trimeric, prefusion F from the other forms tested.Since previously described quaternary-specific antibodies against other class I fusion proteins have in some cases shown preference for the mature protease-cleaved fusion protein over the uncleaved protein , we soug1 to stabilize the weak interprotomer interactions that are normally formed when RSV F is localized in the membrane [Production of soluble prefusion RSV F has thus far relied on the presence of the foldon motif at the C-terminus of Fmembrane , 26. HavAM14 neutralization of RSV is similar in potency to that of the antigenic site Ø antibody D25 , 23. It In our ELISAs, prefusion-specific antibodies D25 and MPE8 both bound to uncleaved postfusion F, albeit with low affinity. One possible explanation is that the presence of pep27 in uncleaved postfusion F leads to a more flexible state in which the association of HRA and HRB is weaker and portions of these epitopes that are normally occluded in the cleaved postfusion state are accessible. This hypothesis is supported by the finding that the six-helix bundle-directed antibody 114F bound tighter to cleaved postfusion F . It is fBesides AM14, other antibodies have been identified that recognize higher-order protein structures in both class I and class II viral fusion proteins. The specificity of these antibodies can be grouped into at least three categories. In the first, antibodies contact only one protomer, but recognize a conformation that exists only in the assembled fusion protein, as is the case for the HIV antibody 35O22, which binds to both gp120 and gp41 subunits . This tyIn addition to quaternary-specific binding, AM14 also exhibited a unique dependence on furin cleavage. RSV F is distinct from the F proteins of other paramyxoviruses due to the presence of two furin sites separated by a glycosylated 27 amino acid spacer, pep27, which is released from the protein after cleavage . There aThe specificity of the potent RSV neutralizing antibody AM14 makes it a useful reagent for probing or isolating the cleaved trimeric state of prefusion F. AM14 may also allow an unparalleled view of prefusion F in its native state, as this antibody can be used to capture trimeric prefusion F without the use of a trimerization motif or stabilizing mutations, as was done for HIV Env using the recently identified trimer-specific antibody PGT151 . AM14 caStrep-Tactin resin (IBA). Tags were removed by digestion with thrombin or HRV3C protease, followed by gel filtration using a Superose 6 column . For crystallization, RSV F proteins were expressed in the presence of kifunensine (5 μM), digested with Endo H (10% w/w), mixed with a 1.5-fold molar excess of Fab, and purified using the Superose 6 column. AviTagged F proteins were biotinylated with biotin ligase BirA (Avidity) and separated from excess biotin by gel filtration with a Superdex 200 column (GE).Plasmids encoding RSV F prefusion (DS-Cav1) and postfusion (F ΔFP) proteins based on strain A2 , 26 werePlasmids encoding antibody heavy and light chains were transfected into Expi293 cells (Invitrogen). IgGs and Fabs were purified using Protein A agarose (Fisher) or CaptureSelect IgG-CH1 affinity matrix (Life Technologies), respectively.Biotinylated proteins were coupled to avidin-coated MagPlex beads at 1 μg per 50,000 beads (Radix). Approximately 1000 beads per well were incubated with 10-fold serial dilutions (1 μM to 0.1 pM) of each antibody in a 384-well plate, washed with PBS plus 0.1% BSA with 0.05% Tween 20 and incubated with 0.33 μg/ml phycoerythrin (PE)-conjugated mouse anti-human IgG Fc secondary antibody (Southern Biotech). Beads were washed and emission at 575 nm was measured using the FLEXMAP 3D flow cytometer (Luminex).Biotinylated DS-Cav1 was immobilized on an SA sensor chip to a total of 292 response units using a Biacore X100 (GE). A buffer-only sample was injected over the DS-Cav1 and reference flow cells, followed by AM14 Fab 2-fold serially diluted from 5 nM to 19.5 pM in HBS-EP+, with a duplication of the 156 pM concentration. The data were double-reference subtracted and fit to a 1:1 binding model using the Biacore X100 analysis software.Samples were diluted to approximately 0.03 mg/ml, adsorbed to a freshly glow-discharged carbon-film grid for 15 sec, and stained with 0.7% uranyl formate. Images were collected semi-automatically using SerialEM  on a FEIAM14 Fab crystals were produced by hanging-drop vapor diffusion by mixing 1 μl of AM14 Fab (8.7 mg/ml) with 1 μl of reservoir solution containing 0.1 M sodium acetate pH 4.5, 0.2 M ammonium sulfate and 25% (w/v) PEG 4000. Crystals were soaked in reservoir solution supplemented with 30% (v/v) ethylene glycol and frozen in liquid nitrogen. Data to 2.0 Å were collected at the MacCHESS beamline .The ternary complex was produced by mixing Endo H-treated DS-Cav1 with a 1.5-fold molar excess each of AM14 Fab and motavizumab Fab before separation of the complex from excess Fab by gel filtration. Crystals were produced by hanging-drop vapor diffusion by mixing 0.67 μl of protein (5.6 mg/ml) with 1.33 μl of reservoir solution containing 11.4% (w/v) PEG 8000, 1.7% (v/v) 2-methyl-2,4-pentanediol and 0.1 M imidazole pH 6.5. Many cryopreservation solutions were tested, but diffraction was highest when the crystal was directly plunged into liquid nitrogen after removal of the cold gas layer . DiffracDiffraction data were processed using the CCP4 software suite: data were indexed and integrated in iMOSFLM  and scalA molecular replacement solution for the 5.5 Å ternary complex was obtained using PHASER with prefusion RSV F (PDB ID: 4JHW ), motavi6 pfu/ml with 3 μg/ml of AM14 for 1 hr prior to infection of confluent HEp2 cells at an MOI of 1.0. Following a 2–3 hour infection, viral inoculum was removed and medium containing 3 μg/ml of antibody was added and incubated at 37°C for 5–7 days. Virus was harvested from wells containing cytopathic effect during the first round of selection and subjected to an additional 2 rounds of selection at 10 μg/ml. Following each round of selection, RNA was isolated from virally infected cells and analyzed by sequencing to determine F protein sequences. After the third round of selection, viruses were plaque purified and microneutralization assays were performed in HEp-2 cells as previously described [RSV strain Long was incubated at 5 x 10escribed .A plasmid encoding RSV F residues 1–574 and harboring the DS-Cav1 stabilizing mutations was created, along with an N426D variant, for expression on the cell surface. Expi293 cells were transfected, harvested 48 hours post-transfection, and washed twice with PBS, followed by incubation with RSV F-specific antibodies (1 μg/ml) for 1 hour at 4°C and Alexa488-conjugated goat anti-human secondary antibody (Invitrogen) (5 μg/ml) for 30 minutes at 4°C. Cells were washed, fixed with 0.5% paraformaldehyde, and evaluated by flow cytometry . Data were analyzed using FlowJo software (Tree Star) and GraphPad Prism.o-phenylenediamine dihydrochloride (Sigma), reactions were stopped with 2 N sulfuric acid and absorbance was read at 490 nm.96-well plates were coated with purified monoclonal antibody  at 6 μg/ml in PBS. Plates were blocked with 2% pig serum in PBS with 0.05% Tween 20 and washed with water. Purified F proteins were serially diluted 4-fold (8 μg/ml to 0.49 ng/ml) and added to plates, which were then washed before incubation with biotinylated anti-HisTag antibody (0.3 μg/ml) (Bio-Rad) and streptavidin-HRP (1:2000) (GE). After addition of Strep-Tactin resins. Separately, DS-Cav1 was produced as described above and mixed with a 1.5-fold molar excess of either AM14 or D25 Fab prior to analysis. Tags were removed from both complexes by thrombin digestion before gel filtration using a Superose 6 XK 16/70 column (GE).RSV F ectodomain consisting of residues 1–513 with a C-terminal thrombin cleavage site, 6x His-tag and Strep-tag II was co-expressed with either AM14 or D25 Fab in FreeStyle 293-F cells. Complexes were purified using Ni-NTA and S1 Fig(A) Neutralization of RSV strains A2, Long, 18537 and 9320 were measured using a microneutralization assay. Two-fold serial dilutions of AM14 (red) or palivizumab (black) were incubated with RSV before infection of HEp-2 cells and detection of RSV F on the surface of infected cells by ELISA. In all strains tested, AM14 was greater than one log more potent than palivizumab. (B) A flow cytometry-based attachment assay was modified from a previously described adherent cell assay , 51. Ant(TIF)Click here for additional data file.S2 FigKD) of 0.07 nM, with association and dissociation rate constants of 1.35 x 106 M-1s-1 and 9.65 x 10-5 s-1, respectively. (B) Binding of D25 to HRA peptide was measured by biolayer interferometry. His-Tagged HRA peptide (RSV A2 F residues 153–211) was immobilized to anti-His sensors and two-fold serial dilutions of D25 IgG from 50 nM to 0.8 nM were assessed for binding on an Octet QK. Red lines are the fit of a global association and then dissociation algorithm in GraphPad Prism, from which the 0.8 nM concentration was excluded. This algorithm was used to calculate an equilibrium dissociation constant (KD) of 1.9 nM for D25 IgG binding to HRA peptide.(A) Binding of D25 Fab to prefusion RSV F was measured by surface plasmon resonance. Prefusion RSV F was immobilized on an SA chip to a total of 300 RU and binding to D25 Fab was measured in 2-fold serial dilutions from 10 to 0.04 nM, with a duplicate of the 0.31 nM concentration. Best fit of the data to a 1:1 binding model is shown in red. D25 Fab bound to immobilized prefusion RSV F with an equilibrium dissociation constant ((TIF)Click here for additional data file.S3 FigStructure of the AM14 Fab with heavy chain shown in blue and light chain shown in white. Complementarity-determining regions (CDRs) are labeled for both the heavy and light chains. The CDR H3 loops of the two Fabs in the asymmetric unit were in different conformations, the second of which is shown in purple. The dotted line indicates a region that was not modeled due to poor electron density. For the complex with RSV F, the Fab with the ordered CDRH3 was used as the search model.(TIF)Click here for additional data file.S4 FigThe asymmetric unit of the ternary complex was composed of one RSV F trimer, three AM14 Fabs and three motavizumab Fabs. The side view and top view of the complex are shown, with RSV F protomers colored tan, light pink and light green. AM14 heavy chains are blue, motavizumab heavy chains are magenta and both AM14 and motavizumab light chains are white.(TIF)Click here for additional data file.S5 FigThe main chains are shown as ribbons within the 2Fo—Fc electron density maps for the refined structure (blue), contoured at 1σ. Only one AM14 Fab and the RSV F trimer are shown for clarity. Chains are colored as in (TIF)Click here for additional data file.S6 FigMolecular surface representations of prefusion F and AM14 Fab colored according to electrostatic surface potential . The surfaces are shown in an open-book representation of the binding interface with the approximate footprint for prefusion F and AM14 Fab outlined in yellow and black. A negatively charged region on the AM14 Fab complements a positively charged region on the lower protomer of prefusion F.(TIF)Click here for additional data file.S7 FigTwo-fold serial dilutions of AM14 (red) or palivizumab (black) were incubated with (A) RSV A Long I79M/R429S/H515N, (B) L160S or (C) N183K before infection of HEp-2 cells and detection of RSV F on the surface of infected cells by ELISA. (D) In a separate assay, two-fold serial dilutions of AM14 were incubated with mKate RSV A2 (grey) or mKate RSV A2 N426D (blue) before infection of HEp-2 cells and measurement of fluorescence (excitation of 588 nm and emission at 635 nm) with a plate reader as previously described . All MAR(TIF)Click here for additional data file.S8 FigPlates were coated with (A) stabilized prefusion F or (B) the N426D mutant (1 μg/ml each) and were washed, blocked and incubated with four-fold serial dilutions of motavizumab (black), D25 (blue), 101F (grey), MPE8 (open blue) and AM14 (red) before detection with HRP-conjugated goat anti-human IgG (Santa Cruz Biotechnology) and Super AquaBlue substrate (eBiosciences Inc.). An ELISA plate reader  was used to read signal at 538 nm. Binding of motavizumab, MPE8 and D25 was similar for both proteins. 101F binding to N426D was slightly reduced, whereas AM14 binding to N426D was reduced approximately 100-fold compared to wild-type prefusion F.(TIF)Click here for additional data file.S9 Fig(A) Coomassie-stained reducing SDS-PAGE gel of the six proteins used in the ELISAs here and in (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.


ADIPOQ) gene and transcription factor 7-like 2 (TCF7L2), but few studies clarified the effect of genetic polymorphisms of ADIPOQ and TCF7L2 on risk of T2DM. We attempted to elucidate association between T2DM and polymorphic variations of both in Taiwan's Chinese Han population, with our retrospective case-control study genotyping single nucleotide polymorphisms (SNPs) in ADIPOQ and TCF7L2 genes both in 149 T2DM patients and in 139 healthy controls from Taiwan. Statistical analysis gauged association of these polymorphisms with risk of T2DM to show ADIPOQ rs1501299 polymorphism variations strongly correlated with T2DM risk (P = 0.042), with rs2241766 polymorphism being not associated with T2DM (P = 0.967). However, both polymorphisms rs7903146 and rs12255372 of TCF7L2 were rarely detected in Taiwanese people. This study avers that ADIPOQ rs1501299 polymorphism contributes to risk of T2DM in the Taiwanese population.Diabetes is a serious global health problem. Large-scale genome-wide association studies identified loci for type 2 diabetes mellitus (T2DM), including adiponectin ( Global prevalence of type 2 diabetes mellitus (T2DM) is 6% but is expected to rise due to aging population ; 2012 saADIPOQ or APM1) and TCF7L2 are located, respectively [ ADIPOQ gene, is an adipose-specific secretory protein involved in many metabolic processes: for example, glucose modulation and fatty acid oxidation [ ADIPOQ are linked with metabolic syndromes: for example, insulin resistance, abdominal obesity, impaired glucose tolerance, dyslipidemia, hypertension, increased fasting glucose [ ADIPOQ showed significant association with obesity and insulin resistance [ ADIPOQ SNPs, rs2241766 and rs1501299, showed significant association with risk of T2DM in the Japanese population [ ADIPOQ gene in T2DM controversial. Any relation between these SNPs and T2DM in the Taiwanese remains unclear. Human transcription factor 7-like 2  [ TCF7L2 gene correlates with T2DM risk and progression in multiple ethnicities [ TCF7L2 and ADIPOQ on T2DM risk is scant in Taiwanese populations; we researched both genes.Genome-wide studies identify diabetes susceptibility loci on chromosomes 3q27 and 10q25.3, where adiponectin genes   is invola. TCF4) , 21. Polnicities . StronglWe recruited 149 individuals with T2DM and 139 healthy controls, as diagnosed by Dr. Ming-Kai Tsai, with basic characteristics of both groups summarized in  ADIPOQ and TCFL2, 33 and 84 SNPs were reported, respectively, from dbSNP database (http://www.ncbi.nlm.nih.gov/SNP). SNPs were based on genetic information for the Chinese (CHB) and the Japanese (JPT) in HapMap (www.hapmap.org). Allele-specific polymerase chain reaction (AS-PCR) analysis was performed with allele-specific primer sets ( ADIPOQ (rs2241766 and rs1501299) and TCF7L2 (rs7903146 and rs12255372). In brief, forward primers specifically complementary to each SNP allele were designed and two PCR reactions were conducted in parallel to determine the allele. PCR used MyCycle thermal cycler (USA) containing 1.25 U of Taq DNA polymerase (TaKaRa), 1X PCR buffer, 200 umol/L dNTP, 100 ng of each primer, and 10 ng of genomic DNA. Amplification was through initial denaturation at 94°C for 5 min: 35 cycles of denaturation at 94°C for 30 s, annealing at 45°C for 30 s, extension at 72°C for 30 s, and final extension at 72°C for 5 min. PCR products were analyzed by 2% agarose gel. We also confirmed accuracy of AS-PCR via Sanger DNA sequencing.Inmer sets  on genothttp://linkage.rockefeller.edu/ott/linkutil.htm) [P value <0.05 constituting statistical significance. Correction for multiple comparisons used positive false discovery rate (FDR) statistics based on the method of Benjamini and Hochberg [Hardy-Weinberg equilibrium (HWE) was tested by online HWE program (til.htm) , and allHochberg , with FDHochberg . ADIPOQ and two SNPs of TCF7L2 genes were genotyped in T2DM patients and controls, using allele-specific polymerase chain reaction analysis ( ADIPOQ and TCF7L2 genotypes are plotted in P = 0.042) in ADIPOQ (rs1501299 (G276T)) polymorphism after Benjamini and Hochberg correction (FDR < 0.05). Yet no differences of genotype frequencies in ADIPOQ (rs2241766 (T45G)) and TCF7L2 (rs7903146) polymorphisms were detected. Surprisingly, genotypes of rs12255372 for TCF7L2 were the only GG genotype appearing in both healthy and T2DM subjects: that is, rs12255372 is not informative among the Taiwanese population. Genotype distributions of all SNPs do not deviate from HWE in either T2DM patients or controls (data not shown).Two SNPs ofanalysis . Distrib ADIPOQ being 28.5% higher among T2DM patients compared to healthy controls  (P = 0.032), with no difference apparent in other polymorphisms. Collectively, these results suggest genetic variation of ADIPOQ, in particular rs1501299 SNP, may have a role in prevalence of T2DM among the Taiwanese population. We likewise compared distribution of genotypes for ADIPOQ rs1501299 SNP among Taiwanese T2DM and control subjects from the current study, plus other ethnic control groups: for example, the European (CEU), the Han Chinese (HCB), and the Nigerian (YRI) groups from HapMap database  . The odddatabase . While G ADIPOQ and TCF7L2 polymorphism in the Taiwanese. Interestingly, significant correlation was noted between ADIPOQ rs1501299 polymorphism and T2DM subjects, with T allele shown as a risk factor for prevalence of T2DM.Novel associated genetic diseases identified by genome-wide association studies are needed for further testing in different populations. Association study is the most important tool for identifying genes conferring susceptibility to complex disorders. Genetic studies on many complex disorders like diabetes and psychiatric traits confirm many risk-associated genetic variants contributing to a subtle effect . Our casPolymorphisms of TCF7L2. TCF7L2 is involved in beta-catenin-dependent Wnt signaling pathway, which employs its physiological functions in pancreatic and intestinal endocrine cells [ TCF7L2 with risk of T2DM in extensive populations [ TCF7L2 polymorphisms rs7903146 and rs12255372 with T2DM prevalence in Taiwan. However, minor allele frequencies of rs7903146 and rs12255372 are 0.023 and unknown, inconsistent with prior study of the Chinese population [ TCF7L2 were rare in Taiwanese population, it is noteworthy to observe linkage between rs7903146 in TCF7L2 and T2DM in ethnic groups [ne cells , 21. Preulations , 32, thopulation . Data hic groups –37. Genec groups . These pPolymorphism of ADIPOQ. Adiponectin, adipocytokine encoded by ADIPOQ, is central to insulin sensitivity and resistance and other metabolic syndrome traits in diverse populations [ ADIPOQ SNPs were linked with plasma adiponectin level among the Caucasian population [ ADIPOQ polymorphisms and T2DM or diabetic nephropathy had also been evaluated from many populations: for example, the European-derived [ ADIPOQ SNP (rs2241766) as prevalent in metabolic syndrome patients versus control groups in the Chinese Han population [ ADIPOQ polymorphisms and T2DM in 149 patients and 139 normal controls in Taiwan (study power = 0.7); genotype frequency of SNP rs2241766 showed no difference between T2DM and controls. Since rs2241766 is a silent mutation for Gly15, this SNP may link with other functional SNPs.ulations , 40. In ulations , 42; ADIpulation , 12 and pulation , 10. ADI-derived , 18, the-derived , 16, 40,-derived , and the-derived  populati-derived . Gao et  ADIPOQ, rs1501299, to find rs1501299 SNP definitely associated with T2DM (P = 0.045). High prevalence of T allele (P = 0.028) and risk of T2DM in the Taiwanese population was observed (P = 0.032), findings that are consistent with studies performed in populations of southern India [We focused on other SNPs ofrn India , Japan [rn India , and Mairn India . Also, Yrn India , which srn India –51, suggrn India . Moreovern India . This poStudy Limitation. One limitation of this study was small sample size due to cost considerations. While genetic analysis of SNP rs1501299 proved significant, we could not entirely exclude a possibility of false positives because of sampling limitations and other factors . Yet we added statistics, FDR < 0.05, as a true positive association to support our conclusions. ADIPOQ and TCF7L2 polymorphism in Taiwan's Han Chinese population: strong correlation emerged between ADIPOQ rs1501299 and T2DM subjects in Taiwan. Taken together, this verifies that ADIPOQ is associated with T2DM risk in Taiwanese population, which may prove useful clinically as a genetic marker. Results suggest ADIPOQ as a key genetic contributor to T2DM that may serve as a clinical biomarker.Our case-control study linked T2DM with