Immune responses to inactivated vaccines against avian influenza are poor due in part to lack of immune memory. Adjuvants significantly increased virus neutralizing titers. We performed comprehensive analyses of polyclonal antibody responses following FDA-approved adjuvanted H5N1-A/Indonesia vaccine, administered in presence or absence of AS03. Using Whole Genome Fragment Phage Display Libraries, we observed that AS03 induced antibody epitope diversity to viral hemagglutinin (HA) and neuraminidase compared with unadjuvanted vaccine. Furthermore, AS03 promoted significant antibody affinity maturation to properly folded H5-HA1 (but not to HA2) domain, which correlated with neutralization titers against both vaccine and heterologous H5N1 strains. However, no increase in heterosubtypic cross-neutralization of Group1-H1N1 seasonal strains was observed. AS03-H5N1 vaccine also induced higher neuraminidase inhibition antibody titers. This study provides insight into the differential impacts of AS03 adjuvant on H5N1 vaccine-induced antibody responses that may help optimize vaccine platforms for future vaccines with improved protection against seasonal and pandemic influenza strains. Adjuvant AS03 improves a bird flu vaccine’s ability to recognize and bind to virion targets. Avian influenza viruses are considered a pandemic threat, and vaccines made from inactivated virus are limited in their efficacy. However, adjuvants such as AS03 have the ability to augment a host’s immunity, prompting Surender Khurana and colleagues from the United States’ Food and Drug Administration to investigate how. In an H5N1 vaccine trial, researchers found that AS03 improved immune responses by increasing the degree, diversity, and intensity of antibody binding to viral surface proteins, which likely caused the observed increase in the neutralization of the virus tested as well as efficacy against other H5N1 strains. The AS03-adjuvanted vaccine did not neutralize seasonal H1N1 influenza strains. This study may inform future vaccine development efforts by partially illuminating the effects of AS03. Furthermore, low pathogenic avian influenza (LPAI) H7N9 viruses have caused five epidemic waves of human infections in China since 2013, and the number of human infections increased significantly during the fifth wave. As of July 26, 2017, there were 1582 laboratory-confirmed human cases of avian H7N9 infection, with 39% fatality rate. In February 2017, highly pathogenic H7N9 viruses possessing multibasic cleavage site in the hemagglutinin (HA) were detected in China.5 Therefore, both avian-derived A/H5N1 and A/H7N9 influenza viruses are considered a pandemic threat due to lack of preexisting immunity in humans. However, inactivated influenza vaccines containing HA and neuraminidase genes from avian influenza reassorted with the A/Puerto Rico/8/34 (PR8) internal genes were found to be poorly immunogenic in human trials, even when the HA content was increased significantly compared with seasonal influenza vaccines.7Humans are mostly immune-naïve to avian influenza strains as they have not been exposed to these strains in their lifetime. Transmission of avian strains from wild birds to poultry with subsequent transmission to humans resulted in high morbidity and mortality among infected individuals and represents pandemic threat. Highly pathogenic (HP) H5N1 viruses still cause significant lethality in bird populations with numerous instances of human transmission resulting in severe human disease with >60% mortality.9 Oil in water adjuvants MF59 and AS03 have been evaluated in multiple preclinical and clinical studies in combination with several influenza vaccines , and were found to significantly augment the humoral immune responses as determined by hemagglutination inhibition (HAI) and microneutralization (MN) assays.13Adjuvants comprised of diverse immune modulators that have been used with vaccines to boost the immune responses by various mechanisms, for example, through upregulating genes in antigen-presenting cells that migrate from the site of vaccination to the draining lymph nodes and promote activation of Th, Tfh, and B cells and cytokine production.15 With the continuous introduction of HP avian influenza strains to humans, rapid evolution and diversification of the H5N1 and H7N9 viruses have highlighted the need for advance stockpiling of H5N1 and H7N9 vaccines, anticipating that vaccination with the stockpiled vaccine can provide at least partial protection until a vaccine matching the pandemic strain becomes available for mass vaccination.19 In addition, stockpiled vaccine could be served to prime recipients for later pandemic-specific vaccination with a single vaccine dose. The AS03-adjuvanted H5N1 A/Indonesia/05/2005 subunit vaccine was approved by the FDA in 2013 and is currently in the US National stockpile.One of the desired attributes of vaccines against avian influenza strains with pandemic potential is the ability to elicit long-term broadly reactive antibodies that could afford at least partial protection against the emerging pandemic strains. Influenza vaccines against avian influenza when combined with oil-in-water adjuvants (MF59 and AS03) generated cross-clade HAI and MN titers and demonstrated improved cross-reactive titers after heterologous prime-boost regiments.24To better understand the adjuvant impact on the humoral immune response to influenza vaccines, we developed several methodologies including whole-genome-phage-display libraries (GFPDL) to probe the epitope repertoires of antibodies elicited in humans by adjuvanted vs. unadjuvanted vaccines. In addition, properly folded HA1 and HA2 domains were used to measure total binding and the dissociation rates of the bound polyclonal antibodies in human polyclonal plasma/serum as correlates of antibody affinity.Since current influenza virus vaccines have demonstrated limited clinical efficacy in recent years, there is an urgent need for next-generation vaccines that can provide broader, long-lasting protection against influenza A and B viruses. It is also expected that adjuvants will be important in the development of the next-generation improved influenza vaccines , that aim to provide cross-subtypic heterologous protection against diverse influenza strains.In the current study, we performed a comprehensive analysis of the antibodies elicited by the FDA-approved AS03-adjuvanted H5N1 (A/Indonesia/05/2005) vaccine by: (1) measuring the diversity of antibody epitope repertoires in HA and NA, (2) evaluating the binding affinity to HA1 vs. HA2 domains, and (3) correlating these results with functional assays (MN and neuraminidase inhibition assay) against homologous and heterologous H5N1 strains. To explore the possibility of oil-in-water adjuvants to augment cross-subtype neutralizing antibodies, we also evaluated the capacity of the AS03-adjuvanted H5N1 vaccine to elicit heterosubtypic (stem-targeting) neutralizing antibodies against H1N1 group 1 virus strains that circulated in the previous decade.The NIH/Center for Human Immunology (CHI) clinical study vaccinated healthy adult volunteers with FDA-licensed monovalent Influenza H5N1 Virus Vaccine (A/Indonesia/05/2005) formulated in combination with or without AS03 adjuvant.p < 0.05), but no differences were observed either earlier (day 21) or later (day 100) time-point postvaccination  with H5N1 A/Indonesia/05/2005 monovalent inactivated vaccine (GSK), containing 3.75 µg HA, either alone or mixed (1:1) with AS03 (oil-in-water) adjuvant Fig. . Serum sThe same serum samples were evaluated for H5N1 cross-clade neutralization. As can be seen in Table 22To further evaluate the impact of the AS03 adjuvant on the quality of the antibody response to H5N1 vaccine, serum samples from a subset of seroconverted subjects enrolled in the NIH/CHI study were pooled at prevaccination and at day 42 (3 weeks after the second vaccination). All prevaccination samples were seronegative (MN titers < 1:20). For the postvaccination serum pooling, samples that had similar MN titers in the unadjuvanted (MN titers: 40−160) and adjuvanted vaccine (MN titers: 160) groups were selected to explore qualitative differences in antibody epitope repertoires following vaccination. Affinity selection was performed on samples using the FLU-GFPDL constructed from HA and NA genes of H5N1 A/Indonesia/5/05 vaccine strain  in both vaccine groups Fig. . Immuniz20  compared with prevaccination sera and fourfold higher than postvaccination samples from the unadjuvanted H5N1 vaccine group  H5N1 vaccine.27 surrounding the RBD. Previously, we demonstrated that sera from H5N1-infected convalescing individuals and human MAbs derived from H5N1 survivors bound to cross-clade conserved conformational epitopes presented on large HA1 fragments identified by panning on GFPDL from the H5N1-A/Vietnam strain.20Neutralization of influenza virus occurs primarily by antibodies recognizing conformational epitopes located on the globular head of the HA molecule23Therefore, it was important to measure the quality of antibodies elicited by the H5N1 vaccine administered with or without AS03 adjuvant, both in terms of titer and avidity of antibodies, using a surface plasmon resonance (SPR)-based real-time kinetics assay to properly folded HA1 globular domain. To that end, purified H5 HA1 protein was produced and characterized for: (1) the presence of oligomers by gel filtration chromatography; (2) functional binding to sialic acid containing glycans in SPR, and (3) binding to red blood cells resulting in hemagglutination, as previously described.As can be seen in Fig. As can be seen in Fig. 22For measurements of antibody affinity maturation, serum samples at 10-fold and 100-fold dilutions were injected on a low density chip surface so as to measure monovalent antibody−antigen interactions of serum antibodies in SPR. Antibody off-rate constants, which describe the fraction of antigen−antibody complexes that decay per second, were determined directly from the serum sample interaction with rHA1 or rHA2 protein using SPR in the dissociation phase as described before.−2/sec) compared to HA1-binding antibodies (10−1/sec) on day 0 (prevaccination). Following vaccination, the affinities of anti-HA2 antibodies were highly variable within groups with no major increase after the second boost. The affinity of HA2 binding antibodies were also similar between the adjuvanted and unadjuvanted groups, except for significant difference observed on day 21 post first vaccination  that replaced the previous H1N1 seasonal strain and continues to circulate in the world today. Most adults have very low or no antibody titers against the old H1N1 strains. Since the H5N1 vaccine strain has a novel head domain compared to H1N1 strains we expected participants in the study to have very low induction of head-specific H1N1 cross-reactive antibodies that can block receptor binding  following H5N1 vaccination, yet be likely to have cross-reactive stem-specific memory B cells that could have been recalled by the AS03-adjuvanted H5N1 vaccine due to the homology between H5 and H1 HA2 stem domain as shown in Fig. 36 MAb CR6261 was used as a positive control in these assays. This stem-targeting MAb was reported to have broad in vitro neutralizing activity against Group 1 H1, H2, H5, H6, H8, H9 influenza subtypes.37 In the modified MN assay, CR6261 neutralized A/New Caledonia/22/09 and A/Solomon Islands/3/06 with endpoint of 0.025–0.05 mg/ml. However, neutralization of A/Brisbane/59/07 was much weaker (>0.1 mg/ml). In addition to the older H1N1 strains, we measured neutralization of H1N1 A/California/04/09, and Gp 2 H3N2 seasonal strain A/Switzerland/9715293/13 . To further explore if the increase in HA2 stem binding antibodies following AS03-adjuvanted H5N1 vaccination led to any increase in stem-targeting neutralizing antibodies, we employed a modified MN assay (3-day) that was previously described by other investigatorsThe prevaccination (day 0) samples from study participants had low neutralizing antibody titers against these H1N1 seasonal influenza strains ranging between <1:10 and 1:160. Postvaccination (day 7) sera samples from both unadjuvanted and adjuvanted groups showed either reduction, no change, or minimal (twofold) increase in neutralization titers, with only two individual samples showing fourfold increases in titers over day 0 against pandemic H1N1 strain in the adjuvanted vaccine group Fig. . Therefo41 It is postulated that induction of NAI titers by a next-generation influenza vaccine could provide better protection against drifted influenza strains. In the FLU-GFPDL analysis, an increase in bound phages containing inserts was observed that mapped to NA, including targets close to the enzymatic site 25 using serum samples from the AS03-adjuvanted vaccine group .Current influenza inactivated vaccines contain varying levels of neuraminidase (NA) in addition to HA, but there is no regulatory requirement to maintain a specific amount of NA per dose. Yet, there is a large body of data supporting the contribution of anti-NA antibodies to protection from severe influenza disease.oup Fig. . To furtTogether, our study demonstrated profound changes in the antibody epitope repertoire and affinity maturation following vaccination with a novel H5N1 immunogen from avian influenza strain with pandemic potential when combined with the oil-in-water adjuvant AS03. The changes in the quality of the antibody responses correlated with an expanded breadth of neutralization against heterologous clades of H5N1, but not against heterosubtypic Group 1 H1N1 influenza viruses.8 For understanding the impact of AS03 adjuvant on vaccination-induced humoral response, samples were collected prevaccination (day 0), 3 weeks post first dose (day 21), 3 weeks post second dose (days 42) and at day 100, for antibody analyses.In the current study, FLU-GFPDL and SPR analyses were used for comprehensive analysis of the humoral responses generated by the FDA-licensed H5N1 A/Indonesia monovalent inactivated vaccine, with or without the AS03 oil-in-water adjuvant.The main findings of the study were: (i) the unadjuvanted low dose vaccine (3.75 µg HA/dose) was poorly immunogenic in terms of eliciting neutralization titers, while the AS03-adjuvanted vaccine was strongly immunogenic leading to 100% seroconversion rates by day 42. Significant cross-clade neutralization of other clade 2 H5N1 strains was also found, with a lower response rate against H5N1 clade 1 virus; (ii) GFPDL and SPR demonstrated H5-binding antibodies in prevaccination sera. These antibodies targeted an epitope within the HA2 domain with a high degree of homology (>90%) to the corresponding sequence in the HA2 of group 1 (H1N1) human seasonal viruses. These HA2 binding antibodies were increased after vaccination with the H5N1 vaccine in both unadjuvanted and adjuvanted vaccine groups; (iii) vaccination with AS03-adjuvanted H5N1 vaccine (but not with unadjuvanted vaccine) induced epitope spreading from HA2 to HA1 (including large fragments spanning the RBD) resulting in a sevenfold increase in the ratio of HA1/HA2 bound inserts compared to unadjuvanted vaccination; (iv) SPR real-time kinetic analyses with HA1 and HA2 domains revealed a gradual increase in the total binding and affinity of antibodies targeting the globular HA1 domain primarily in recipients of the AS03-adjuvanted H5N1 vaccine; (v) a strong inverse correlation was found between the anti-HA1-binding antibody off-rates (surrogate of antibody affinity) and the neutralization titers against either A/Indonesia (vaccine strain) or the other clade 2 viruses. In contrast, no clear correlation was found between HA2 binding titers (or off-rates) and virus neutralization titers; (vi) expansion of binding to targets in the neuraminidase protein (NA), including sites close to the sialic acid binding enzymatic site was also observed for sera from AS03-adjuvanted vaccine recipients using GFPDL. In agreement, the increase in neuraminidase inhibition (NAI) titers was more pronounced in this group; (vii) no increase in heterosubtypic neutralization of Group 1 H1N1 seasonal influenza viruses was found in either H5N1 vaccine group.43 It is likely that the main reason for this difference is the vaccine dose. The unadjuvanted H5N1-A/Vietnam vaccine contained a 90 µg HA/dose, which is sixfold higher than the antigen dose in seasonal vaccine (15 µg HA) and 24-fold higher than the antigen dose of H5N1-A/Indonesia (3.75 µg HA) that was approved by the FDA in combination with AS03 used in the current study. Therefore, the addition of AS03 provided a significant dose-sparing effect that will be of great value in the case of mass global vaccination. Similar to the finding with H5N1 immunogens, several clinical trials conducted recently demonstrated improved immunogenicity and dose sparing of H7N9 vaccines when combined with AS0345 and with MF59.45The response rate against the unadjuvanted H5N1-A/Indonesia vaccine in the current study was lower than that reported against H5N1 A/Vietnam in the earlier vaccine trial that supported licensure of the monovalent inactivated vaccine from Sanofi Pasteur.46 The H1N1 strains used included A/New Caledonia/1999, A/ Solomon Islands/2006, and A/Brisbane/59/2007, which circulated in the US in the years 1999–2005, 2006/7, and 2007–2009, respectively. Since H5 and H1 globular heads share low homology, any increase in anti-H1N1 neutralization titers between day 0 and day 7 post vaccination should reflect a recall of HA2 stem-targeting neutralizing antibodies. However, we found no clear increase in neutralization titers against the three H1N1 strains, in spite of low prevaccination titers against these seasonal H1N1 influenza strains. Therefore, the AS03-adjuvanted H5N1 vaccine used in our study (containing a novel HA1 head domain) did not fulfill the requirements for “A Universal Influenza Vaccine” as outlined in a recent white paper.32 One of the limitations to this conclusion is the fact that currently there is no established correlate of protection for stem-based influenza vaccines. In a previous study, passive transfer of antibodies from H5N1-vaccinated individuals  protected mice from challenge with a chimeric H9/1 N3 strain.33 It was postulated that Fc-mediated functions play a key role in such protection.47 Multiple in vitro and in vivo assays based on the antibody Fc functions have been described including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis, and antibody-dependent cell-associated viral elimination. To date, no one assay has been fully predictive of in vivo protection against heterotypic challenge viruses in humans and animal models.55 Furthermore, the potential of non-HAI stem-targeting antibodies to enhance lung pathology after infection, in absence of head-targeting antibodies, should be carefully evaluated in preclinical models.59Since the HA2 sequence is conserved among Group 1 H5N1 and H1N1 strains, it has been hypothesized that vaccination with H5N1 in adult populations that have been exposed to seasonal influenza strains, either due to seasonal influenza infection or vaccination will recall HA2 stem-specific antibodies that can cross-protective Group 1 H1N1 strains; this is the basis for development of stem-based next-generation influenza vaccines. It is postulated that adding an adjuvant like AS03 will significantly enhance the magnitude of such HA2-specific recall response early after vaccination with novel head containing vaccines such as those for H5N1. Indeed, we found an increase in HA2 binding antibodies on day 21 post first vaccination using both GFPDL and SPR. Therefore, we evaluated the possibility that soon after vaccination broad heterosubtypic antibodies could be detected in the sera specific for the more conserved HA2 stem domain, by measuring neutralization of H1N1 strains that circulated in the US a decade ago. A modified neutralization assay (72 h) with increased sensitivity to detect stem-targeting broadly neutralizing antibodies was used.60 In a separate study, subjects receiving AS03-adjuvanted H1N1pdm0946 showed an increase in both anti-head and anti-stem antibodies. A significant increase in HI titers against the homologous strain was found in both previously vaccinated  or unvaccinated individuals . In contrast, a very modest increase in neutralization of a chimeric virus expressing cH9/1N1 and ADCC (using cH6/1 HA-coated plates and CD16+ NK cell line) (<2 fold) was reported.46 Therefore, the conclusions of that study regarding heterosubtypic immunity are not significantly different from our study using AS03-adjuvanted avian influenza strain (H5N1 A/Indonesia) vaccine.Previous studies in humans using AS03-adjuvanted H1N1pdm09 vaccine combined with B-cell receptor (BCR) sequencing of isolated of plasma blasts on day 7 provided evidence for improved recruitment of total antibody secreting cells, that included naïve (unmutated BCR) B cells and recalled preexisting memory B cells in the adjuvanted group compared to the unadjuvanted group.61 It has been suggested that NA should be a component of next-generation influenza vaccines since NA-inhibiting antibodies were shown to correlate with protection from severe influenza disease.55 In the current study, AS03-adjuvanted H5N1 vaccine promoted an expansion of the anti-NA epitope repertoire in serum antibodies  H5N1 vaccine, suggesting recruitment of naïve B cells with BCRs targeting important protective sites in the HA1 globular domain and the NA proteins contained in the H5N1 vaccine. Future influenza vaccines that incorporate both HA and NA components are likely to benefit from oil-in-water adjuvants such as AS03 and similar platforms.While adjuvants in combination with traditional influenza vaccines  have not yet provided the breadth and duration of protection required from a universal influenza vaccine, certain adjuvants most likely will be required for optimal and sustainable immune responses against the next-generation influenza vaccines. Thus, an iterative approach for selection of the most promising immunogen design, and the optimal adjuvant will be an important component in the development of this new generation of influenza vaccines.Serum samples from H5N1 human vaccine trial were obtained from the Center for Human Immunology (CHI)-sponsored A/Indonesia/5/05 vaccine trial using the FDA-approved AS03-adjuvanted H5N1 (A/Indonesia/05/2005) vaccine conducted at NIAID, NIH. The study protocol was approved by the NIH Institutional Review Board and the FDA . A written informed consent was obtained from all participants. All samples were de-identified. The study samples were coded and all the antibody assays were performed blindly. After the data were generated, samples were unblinded to perform the data analysis in this study. The protocols were evaluated by CBER/NIH Research Involving Human Subjects Committee and were conducted under RIHSC exemption #03-118B.21 fSK-9-3 is a gIIIp display-based phage vector where the desired polypeptide can be expressed as a gIIIp fusion protein. The phage display libraries used in the current study expressed inserts spanning the HA and NA genes (referred as HA-NA).The phage display libraries expressing inserts spanning the HA and NA genes of the H5N1-A/Indonesia/05/2005 strains were constructed as previously described for H5N1 A/Vietnam/1203/04 GFPDL.21For each round of panning, an equal volume of sera was used for each group. Prior to panning of GFPDL with polyclonal human antibodies, serum components, which might nonspecifically interact with phage proteins, were removed by incubation with ultra violet-killed M13K07 phage-coated Petri dishes. Subsequent GFPDL selection was carried out in solution (with Protein A/G-Sepharose) and inserts of bound phages were PCR amplified and sequenced as previously described.NotI-PacI inserts into a T7 promoter-based pSK expression vector in which the desired polypeptide can be expressed as a fusion protein with His6 tag at the C-terminus. The HA1 and HA2 proteins were expressed and purified as described before.23 Briefly, E. coli Rosetta Gami cells (Novagen) were used for expression of H5N1 proteins. Following expression, inclusion bodies were isolated by cell lysis and multiple washing steps, denatured, refolding in redox folding buffer and dialyzed. The dialysate was filtered through a 0.45 µm filter and was subjected to purification by HisTrap Fast flow chromatography. The purified proteins were characterized for presence of oligomers by Gel filtration chromatography and functional binding to RBC in a hemagglutination assay.The DNA gene segments corresponding to the HA1 and HA2 proteins of H5N1-A/Indonesia/05/2005 and H5N1-A/Vietnam/1203/2004 were cloned as 22 The recombinant HA globular domain (rHA1-His6) or HA stalk domain (rHA2-His6) for the A/Indonesia/05/2005 (clade 2.1) or from H5N1- A/Vietnam/1203/2004 influenza virus strain was coupled to a GLC sensor chip with amine coupling with 100 and 500 resonance units (RU) in the test flow cells. Samples of 60 µL sera at 10-fold and 100-fold dilutions were injected at a flow rate of 50 µL/min (120-s contact time) for association, and disassociation was performed over a 600-s interval (at a flow rate of 50 µL/min). Responses from the protein surface were corrected for the response from a mock (no coating) surface and for responses from a separate, buffer only injection. Binding kinetics for the human vaccine sera and the data analysis were calculated with Bio-Rad ProteOn manager software (version 3.0.1). Antibody off-rate constants, which describe the fraction of antigen−antibody complexes that decay per second, were determined directly from the serum/plasma sample interaction with rHA1 or rHA2 protein using SPR in the dissociation phase and calculated using the Bio-Rad ProteOn manager software for the heterogeneous sample model as described before.22 Off-rate constants were determined from two independent SPR runs.Steady-state equilibrium binding of post-H5N1-vaccinated human sera was monitored at 25 °C using a ProteOn surface plasmon resonance biosensor (Bio-Rad).Viral-neutralizing activity was analyzed in an MN assay in MDCK cells based on the methods of the pandemic influenza reference laboratories of the Centers for Disease Control and Prevention (CDC) with minor modifications provided in the updated SOP issued by the CDC. Antibody-neutralization titers by MN were measured against H5N1 vaccine strains of A/Vietnam/1194/2004 (clade 1), A/Indonesia/5/2005 (clade 2.1), A/Turkey/15/2006 (clade 2.2), A/Egypt/3072/2010 (clade 2.2), and A/Anhui/1/2005 (clade 2.3.4) as well as the H1N1pdm09 . Sera were tested at an initial dilution of 1:20, and those that were negative (<1:20) were assigned a titer of 10. All sera were tested in triplicate, and the geometric mean value was used for analysis.34 MDCK cells (2×105/100 µL/well) were seeded in flat bottom 96-well plates and incubated at 37 °C overnight. The following day, twofold serially diluted RDE-treated serum samples (starting at 1:10) were added to a separate 96-well tissue culture plate in duplicates. Diluted sera were incubated with 100 TCID50/100 µL/well influenza virus  for 1 h at room temperature with shaking. Cell-containing 96-well plates were washed with PBS, and 200 µL/well of the virus-sera mixture was added and incubated at 37 °C for 72 h. Plates were centrifuged at 3000 rpm for 5 min and 50 µL of the supernatant was added to 50 µL of 1% human red blood cells in a U-bottom 96-well plate. Agglutination was read after incubation for 60 min at 37 °C.For measuring cross-subtypic neutralization activity against H1N1 and H3N2 seasonal influenza strains that is more sensitive to neutralizing activity due to stem-specific antibodies, a 3-day infection assay was performed.42 To prevent false NAI signals due to interfering HA antibodies, virus reassortants that combine H5N1 NA and a mismatched HA of the H6 subtype was generated by reverse genetics as previously described62 to measure H5N1-specific NA activity inhibiting antibody titers.The enzyme-linked lectin assay (ELLA) was used for the detection of NA-inhibiting antibodies in immune antisera as previously described.p values) were examined for statistical significance by the multiple comparison adjustment using the Bonferroni method. In addition to p values, the 95% confidence intervals of the differences between study groups are also provided. A p value less than 0.05 was considered significant. Analysis was conducted without alpha adjustment for multiplicity. Spearman correlations are reported for the calculation of correlations between off-rate and MN titers combined across all vaccine groups. All statistical calculations were performed using ANOVA.Differences between groups (The data sets generated during and/or analyzed during the study are available from the corresponding author upon reasonable request.Supplementary Figure 1


Highly pathogenic avian influenza (HPAI) H5N1 and H5N8 have become endemic among domestic poultry in Egypt since 2006 and 2016, respectively. In parallel, the low pathogenic avian influenza H9N2 virus has been endemic since 2010. Despite the continuous circulation of these subtypes for several years, no natural reassortant has been detected so far among the domestic poultry population in Egypt. In this study, the HPAI (H5N2) virus was isolated from a commercial duck farm, giving evidence of the emergence of the first natural reassortment event in domestic poultry in Egypt. The virus was derived as a result of genetic reassortment between avian influenza viruses of H5N8 and H9N2 subtypes circulating in Egypt. The exchange of the neuraminidase segment and high number of acquired mutations might be associated with an alteration in the biological propensities of this virus. Highly pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 viruses have become endemic among domestic poultry in Egypt since 2006 and 2010, respectively ,4, whereOn 31 December 2018, twenty oropharyngeal and cloacal swabs were collected from a commercial 90-days-old Muscovy duck farm (of 5000 birds) at Mansoura city, Dakahlia governorate, in Egypt. Samples were taken prior to slaughtering as a part of an active surveillance program conducted by the National Laboratory for veterinary quality control on poultry production (NLQP) and General Organization for Veterinary Services. Ducks were vaccinated via an H5N1 vaccine at 7 days old. Ducks were apparently healthy showing no signs of disease. Collected samples were submitted to NLQP for virus identification and isolation. All experiments in this study were conducted in accordance with the ethically approved protocol (AHRI-18032019) of the Animal Health Research Institute, Giza, Egypt.Viral RNA was extracted from the pooled samples using the QIAamp Viral RNA Mini Kit  according to the manufacturer’s instructions. Pooled samples were tested using one step Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)  for the M gene of influenza type A viruses  using thVirus isolation was performed via inoculation into the allantoic cavity of 10-day-old specific pathogen free (SPF) embryonated chicken eggs (ECE) according to the World Organization for Animal Health (OIE) diagnostic manual . Collect® Taq DNA Polymerase  with primers described previously [The complete genome sequences of the Egyptian virus A/duck/Egypt/VG1099/2018 (EG-VG1099) was amplified using RT-PCR with SuperScript-III One-Step RT-PCR System with Platinumeviously ,13. The ® software, version 11.0.5 [http://tree.bio.ed.ac.uk/software/figtree/).The obtained sequences were assembled and edited using the Geneiousn 11.0.5 . A Basicn 11.0.5 . Phylogen 11.0.5 . Trees wPooled samples were found positive for the influenza A H5N2 subtype and negative for H7 and H9, as well as N1 and N8. Ducks were apparently healthy with neither disease symptoms nor abnormal mortalities. The virus was isolated, confirmed, and named A/duck/Egypt/VG1099/2018 (EG-VG1099).The complete genome sequences of the Egyptian virus EG-VG1099 were obtained and sequences generated in this study were submitted to the Global Initiative on Sharing All Influenza Data (GISAID) platform under the accession number: EPI1387245-52. Nucleotide identity analysis showed that the EG-VG1099 virus was closely related, according to its hemagglutinin (HA) gene segment, to HPAI H5N8 viruses isolated in Europe 2016–2017, with a nucleotide sequence identity of 98% to the A/Eur_Wig/NL-Greonterp/16015653-001/2016; the same identity of 98% with A/duck/Egypt/F446/2017(H5N8) virus was also recorded. The neuraminidase (NA) gene of the EG-VG1099 virus shared 97% nucleotide identity with the Egyptian LPAI H9N2 virus A/chicken/Egypt/D10700/2015(H9N2) , and no Phylogenetic analysis of the HA gene segment showed that the EG-VG1099 virus was closely related to HPAI H5N8 viruses reported in Europe during 2016–2017 , and phylogenetically distinguished from the previously reported HPAI H5N8 viruses in Egypt A. The NAThe HA gene of the EG-VG1099 virus possessed multiple basic amino acids, “PLREKRRKR/GLF”, in the cleavage site of the HA indicating high pathogenicity of this virus. The EG-VG1099 virus exhibited R76S, S98R, A138S, A160V, and R173Q amino acid substitutions (H3 numbering) at its HA protein, distinguishing it from previously reported HPAI H5N8 viruses of clade 2.3.4.4 (group B). Amino acid substitutions at positions S98R, A138S, and A160V have been reported previously to be related to virulence and host specificity . The NA Currently, Egypt faces endemic co-circulation of HPAI H5N1, H5N8) and LPAI (H9N2) viruses, where a simultaneous detection of the three subtypes has been detected . The co-, H5N8 ann = 12) in the NA have been noticed. Only positions 143 and 344 are located in the antibody recognition sites of the NA coded protein and have been reported in the literature to be related to the antigenic drift/escape mutant [Further, different amino acid substitutions associated with either virulence or host adaptation have been observed in the newly detected Egyptian HPAI H5N2 virus (EG-VG1099). Amino acid substitutions  in the antibody recognition sites of the HA have been observed. These mutations were described to enhance the binding of AIV H5 with the α2.6 sialic acid receptor and increase virulence in the mammalian animal model ,18. Furte mutant ,20. HoweMoreover, our study underlines the importance of active surveillance in the timely detection of new AIV subtypes. It can be expected that continuing surveillance activities might lead to the detection of additional new cases; hence, surveillance should be strengthened with special emphasis in the surrounding regions of the outbreak. Interventions, like improved biosecurity practices in poultry production enterprises, and live bird trading and marketing practices in Egypt, should be considered to reduce the dissemination of AIV and reduce the chance of reassortment raised through co-infection.


The lauroyl G3 conjugates showed the highest toxicity amongst the PAMAM dendrimer conjugates investigated and were significantly more toxic than the lauroyl-G0-naproxen conjugates. The permeability of naproxen across monolayers of Caco-2 cells was significantly increased by its conjugation to either G3 or G0 PAMAM dendrimers. Lauroyl-G0 conjugates displayed considerably lower cytotoxicity than G3 conjugates and may be preferable for use as a drug carrier for low soluble drugs such as naproxen.The present study compares the use of high generation G3 and low generation G0 Polyamidoamine (PAMAM) dendrimers as drug carriers of naproxen (NAP), a poorly water soluble drug. Naproxen was conjugated to G3 in different ratios and to G0 in a 1:1 ratio via a diethylene glycol linker. A lauroyl chain (L), a lipophilic permeability enhancer, was attached to G3 and G0 prodrugs. The G3 and G0 conjugates were more hydrophilic than naproxen as evaluated by the measurement of partitioning between 1-octanol and a phosphate buffer at pH 7.4 and pH 1.2. The unmodified surface PAMAM-NAP conjugates showed significant solubility enhancements of NAP at pH 1.2; however, with the number of NAP conjugated to G3, this was limited to 10 molecules. The lactate dehydrogenase (LDH) assay indicated that the G3 dendrimer conjugates had a concentration dependent toxicity towards Caco-2 cells. Attaching naproxen to the surface of the dendrimer increased the IC Dendrimers represent highly branched macromolecules that have a well-defined structure with precisely controlled size and shape as well as terminal group functionality . The potIn dendrimer-drug conjugates, the drug is combined through a covalent bond either directly or via a linker/spacer to the dendrimer. Our previous work showed that the direct conjugation of a drug to PAMAM dendrimer (via an amide bond) resulted in a stable conjugate that would not be suitable for use as a drug delivery system . In contIn the present study, naproxen  is conju1H-NMR and 13C-NMR spectroscopy [The synthesis and characterization of G0 dendrimer conjugates were previously reported ,11 in a troscopy . G3 dendG3-(deg-NAP)x and LyG3-(deg-NAP)x were purified by size exclusion chromatography, solubilization in water  and dialysis (MW cutoff = 3500 Da) for 48 h.1H-NMR spectroscopy of the conjugates showed the presence of NAP-deg and/or lauroyl chain and G3 PAMAM dendrimer in the product ) of naproxen and conjugates between 1-octanol and phosphate buffer (pH 7.4) were determined as an indicator reflecting the change in the lipophilicity of naproxen after attaching to G0 or G3 PAMAM dendrimers. The conjugation of naproxen to both G0 and G3 dendrimers resulted in a significant decrease in the lipophilicity of naproxen as indicated by the apparent partition coefficients of dendrimer naproxen prodrugs (p < 0.05) lower than that of naproxen) as a result of the presence of lipophilic chains (lauroyl chains) on the surface of dendrimer (p > 0.05) in lipophilicity compared to the unmodified surface conjugates. Apart from Naproxen (pKa 4.2) which is ionised at pH 7.4, there was no significant difference in log Kapp(o/w) values of the conjugates at both pH 7.4 and pH 1.2.Solubility is one of the physicochemical parameters that influence drug permeability, and therefore its pharmacokinetic profile and therapeutic activity. In order to increase the solubility of lipophilic naproxen, it was attached to the surface groups of highly water soluble PAMAM dendrimer through a degradable linkage . Since dprodrugs . For G0 endrimer . In the p < 0.05) in its solubility. The highest solubility of conjugated naproxen is shown for the conjugates that are not surface modified  resulted in significant enhancements  and p < 0.05) conjugate cytotoxicity (L6G3-(deg-NAP)5) and an even more pronounced increase (p < 0.05) in cytotoxicity was found when 12 lauroyl chains were attached (L12G3-(deg-NAP)5), which was excluded from transport studies due to its high cytotoxicity. These results may be explained by the intrinsic cytotoxicity of the lauroyl chain [50 of LG0-deg-NAP and that of G3 PAMAM dendrimer conjugates shows that the G0 prodrug is significantly less toxic (p < 0.05) than G3 and its conjugates at the same concentrations . The concentration of conjugates used in the study were all equivalent to 100 µM naproxen . Approxip < 0.05) than that of naproxen itself. No significant difference (p > 0.05) can be observed between the transport of G3 conjugates (G3-(deg-NAP)5 and G3-(deg-NAP)10) and G0-deg-NAP. These results suggest that the PAMAM dendrimer functions as a drug carrier that is able to cross cellular barriers and enhance the permeability of the drug regardless of the generation. Moreover, these findings are in agreement with previous observations that low generation PAMAM dendrimers (G0 and G1) exhibit significantly less cytotoxicity and higher permeability than higher generations  [The transport of G3-(deg-NAP)5 and G3-(deg-NAP)10 across Caco-2 monolayers, especially in the A→B direction, was similar and both were significantly higher ( and G4) . A comparison between the transport of naproxen-diethylene glycol (NAP-deg) and naproxen confirms that the enhanced permeability of the conjugate arises from the attachment of the drug to the PAMAM dendrimer. The high permeability of PAMAM dendrimer conjugates may be explained by the presence of positively charged amine surface groups. Knipp et al.  reportedp < 0.05) in the transport of naproxen across epithelial cells (p < 0.05) in both directions across Caco-2 cell monolayers of all the PAMAM dendrimer conjugates evaluated. However, L-G0-deg-NAP showed an appreciable enhancement of naproxen transport (p < 0.05) compared to L6G3-(deg-NAP)5 5 . In gene® 12 mm diameter) and cluster plates (96 well) were purchased from Corning Costar UK . Naproxen-diethylene glycol (NAP-deg), NAP-deg-(4-nitrophenyl carbonate), lauroyl (4-nitrophenyl carbonate), G0-diethylene glycol-naproxen (G0-deg-NAP) and lauroyl-G0-diethylene glycol-naproxen (L-G0-deg-NAP) were synthesized and characterized as previously described [13C and 1H-NMR spectroscopy . 13C-NMR spectra were assigned with the aid of DEPT-135. HPLC analyses were carried out using a Hewlett-Packard Series II 1090  instrument equipped with a Luna 5 µm, C18 column . The solvent system for characterization of the conjugates was methanol:ACN:H3PO4 (0.05% w/v) (10:32:58) for 2 min then (48:32:20) for the remaining elution time. The flow rate was 1.2 mL/min and UV detection was at λ = 230 nm. HPLC solvent systems for the apparent partition coefficients and transport studies: methanol:aq H3PO4 (0.05% w/v) (80:20) using phenanthrene as an internal standard. The flow rate was 1.2 mL/min and UV detection was at λ = 230 nm.Third generation PAMAM dendrimers (G3) with ethylenediamine cores were purchased from Dendritech Inc. . Trifluoroacetic acid (TFA), triethylamine (TEA), (S)-(+)-6-methoxy-α-methyl-2-naphthaleneacetic acid (naproxen), 4-nitrophenyl chloroformate, 1-dodecanol , ethylenediamine tetraacetic acid (EDTA), lactate dehydrogenase assay kits, trypan blue, Sephadex 25, and Sephadex LH-20 were purchased from Sigma-Aldrich Co. Ltd. . Cell culture materials were from Gibco BRL Life Technologies . Polycarbonate cell culture inserts  (1 mL), 10% excess of the required amount of NAP-deg-(4-nitrophenyl carbonate) was added dropwise over 2 hours (h) to a stirred solution of G3 dendrimer in DMF (2 mL). The reaction mixture was stirred for 5 days. DMF was evaporated under vacuum. The residue was dissolved in 3 mL water and filtered. The filtrate was purified by size exclusion chromatography, using Sephadex 25, eluting with methanol:water (1:5). G3-(deg-NAP)5 and G3-(deg-NAP)10 were synthesized and characterized by v/v). The resulting product was dissolved in water and filtered. The filtrate was concentrated under vacuum and purified again by size exclusion chromatography using Sephadex 25, eluting with methanol:water (1:5 v/v). L6G3-(deg-NAP)5, L12G3-(deg-NAP)5, were synthesized and characterized by 1H-NMR and 13C-NMR spectroscopy . In order to yield the target ratio L:G3 in 1 mL DMF, 10% excess of the required amount of lauroyl (4-nitrophenyl carbonate) was added dropwise over 2 h to a stirred solution of G3-(deg-NAP)5 in DMF (2 mL). The reaction mixture was stirred for 5 days. DMF was evaporated under vacuum and the residue was purified by size exclusion chromatography using Sephadex LH 20 with methanol:water (5:1 app(o/w)) of G0 and G3 dendrimer conjugates between 1-octanol and phosphate buffer (pH 7.4) and pH 1.2 (0.06 M hydrochloric acid buffer) were determined at 37 °C. Before use, the 1-octanol was saturated with phosphate buffer for 24 h by stirring vigorously. A known amount of conjugate in phosphate buffer  was shaken for 72 h with 1-octanol (5 mL) to achieve equilibrium, and the phases were separated by centrifugation at 10,000 revolution per minute (rpm) for 5 minutes (min). All experiments were performed in triplicate. The concentrations of the compounds in the buffer phase before and after partitioning were determined by HPLC . The apparent partition coefficients (KThe solubility of G0 and G3 dendrimer conjugates in pH 1.2 (0.06 M hydrochloric acid buffer) were determined at 37 °C. All synthesized amount of conjugate (50 mg) added to the buffer  was shaken for 72 h to achieve saturation. The unsolubilised conjugate was then filtered . All experiments were performed in triplicate. The concentrations of the compounds in the buffer were determined by HPLC.2 and 95% relative humidity in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% foetal bovine serum, 1% non-essential amino acids, 50 IU/mL penicillin. and 50 mg/mL streptomycin. After 24 h the medium was removed and the cells were washed in phosphate buffered saline and 200 µL Hanks Balanced Salt Solution (HBSS) containing G3 PAMAM dendrimer, conjugates or G0 PAMAM dendrimer at different concentrations  (passage 110–120) were seeded at 10,000 cells/well in 96-well plates and maintained at 37 °C in an atmosphere of 5% COr weight  for eachThe percentage was calculated using the following equation:5 cells/cm2. Cells were grown at 37 °C in an atmosphere of 5% CO2 and 95% relative humidity in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum, 1% non-essential amino acids, 50 IU/mL penicillin and 50 mg/mL streptomycin. The medium was changed on alternate days for 21–22 days. The integrity of cells was assessed by measuring the transepithelial electrical resistance (TEER) using a voltohmmeter  before and after the experiments. Prior to transport experiments, cells were equilibrated with HBSS for 20 min at 37 °C, and the TEER was determined. The TEER value, corrected for the blank filter resistance, was in the range of 800–950 Ω·cm2. Only confluent monolayers were used for the transepithelial transport studies. Transport of compounds was determined in both apical-to-basolateral (A→B) and basolateral-to-apical (B→A) directions. The transport medium (TM) was HBSS with 25 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) and was placed in the donor and receiver compartments. Naproxen, G0 or G3 dendrimer conjugates  were seeded onto polycarbonate 12-well Transwell filters  statistical test was performed to allow comparison of more than two groups and Student’s The synthesis, characterization, cytotoxicity and transport studies of G3 PAMAM dendrimer-naproxen conjugates using diethylene glycol as spacer/linker are reported. Partition coefficient results indicate that G3-naproxen conjugates were significantly more hydrophilic than the parent drug. A comparison was made between the cytotoxicity and transport profiles of G3 dendrimer conjugates and that of G0 dendrimer conjugates. Cytotoxicity studies showed that G0 dendrimer conjugates were only slightly toxic towards Caco-2 monolayers, and their toxicity was significantly lower than that of G3 dendrimer conjugates. Conjugation of naproxen to G0 and G3 PAMAM dendrimer significantly increased its permeability in both directions across Caco-2 monolayer. A more pronounced increase of naproxen transport was observed when lauroyl chains were used as surface modifiers. No significant difference was shown between the transport profiles of G3 and G0 PAMAM conjugates. However, surface modified G3 dendrimer conjugates showed a higher permeability and significantly higher cytotoxicity than that of surface modified G0 dendrimer conjugates. Future investigations may focus more on the impact of conjugation on the density of surface charge for dendrimer conjugates and the aggregation behaviour of these conjugates in aqueous solutions. Overall, our results suggest that G0 PAMAM dendrimers demonstrate potential as nanocarriers for the enhancement of oral bioavailability of naproxen as a model for low aqueous solubility drugs.


SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated its composition in Drosophila melanogaster. Using transgenic flies that exclusively express Flag-tagged SMN from its native promoter, we previously found that Gemin2, Gemin3, Gemin5, and all nine classical Sm proteins, including Lsm10 and Lsm11, co-purify with SMN. Here, we show that CG2941 is also highly enriched in the pulldown. Reciprocal co-immunoprecipitation reveals that epitope-tagged CG2941 interacts with endogenous SMN in Schneider2 cells. Bioinformatic comparisons show that CG2941 shares sequence and structural similarity with metazoan Gemin4. Additional analysis shows that three other genes  are not orthologous to Gemins 6-7-8, respectively, as previously suggested. In D.melanogaster, CG2941 is located within an evolutionarily recent genomic triplication with two other nearly identical paralogous genes (CG32783 and CG32786). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability.Spinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human  Despite in SMN1 . AdditioTo date, no definitive link has been established between a specific function of SMN and SMA pathogenesis. Moving forward, the key question in the field is to identify which of the many potential activities of SMN lie at the root of the neuromuscular dysfunction. Given that SMA is a spectrum disorder with a broad range of phenotypes .in vitro transcribed Sm-class snRNAs, purified recombinant SMN and Gemin2 are sufficient for Sm core assembly activity  and STRAP  are peripheral members of the SMN complex and may thus serve in a regulatory capacity. In support of this idea, STRAP/UNRIP is found in a separate complex with a cap-independent translation factor called UNR  was originally thought to contain only Gemin2 and Gemin3 (rigor mortis (Rig) encodes a potential metazoan Gemin5 ortholog, but Gemin5/Rig protein failed to co-purify with SMN in Schneider2 (S2) cells Gaulos ( is orthologous to a metazoan SMN complex protein (Gemin4). Using comparative genomic analysis, we conclusively demonstrate that HezronCG14164), Sabbat (CG31950)( and CG2371)Valette .Embryonic lysates were prepared by crushing the animals in lysis buffer  with 1X protease inhibitor cocktail (Invitrogen) and clearing the lysate by centrifugation at 13,000 RPM for 10 min at 4°. S2 cell lysates were prepared by suspending cells in lysis buffer  with 10% glycerol and 1x protease inhibitor cocktail (Invitrogen) and disrupting cell membranes by pulling the suspension through a 25 gauge needle (Becton Dickinson). The lysate was then cleared by centrifugation at 13,000 RPM for 10 min at 4°. Cell fractionation was performed using a standard protocol  for 2h-ON at 4C with rotation. The beads were washed with RIPA lysis buffer or three times and boiled in SDS gel-loading buffer. Eluted proteins were run on an SDS-PAGE for western blotting.Drosophila embryos were collected from Oregon-R control and Flag-SMN flies, dechorionated, flash frozen, and stored at -80°. Embryos (approx. 1gr) were then homogenized on ice with a Potter tissue grinder in 5 mL of lysis buffer containing 100mM potassium acetate, 30mM HEPES-KOH at pH 7.4, 2mM magnesium acetate, 5mM dithiothreitol (DTT) and protease inhibitor cocktail. Lysates were centrifuged twice at 20,000 rpm for 20min at 4° and dialyzed for 5h at 4° in Buffer D . Lysates were clarified again by centrifugation at 20000 rpm for 20 min at 4C. Lysates were flash frozen using liquid nitrogen and stored at -80C before use. Lysates were then thawed on ice, centrifuged at 20000 rpm for 20 min at 4C and incubated with rotation with 100 uL of EZview Red Anti-FLAG M2 affinity gel (Sigma) for 2h at 4C. Beads were washed a total of six times using buffer with KCl concentrations ranging from 100mM to 250mM with rotation for 1 min at 4° in between each wash. Finally, Flag proteins were eluted 3 consecutive times with one bed volume of elution buffer  containing 250ug/mL 3XFLAG peptide (sigma). The eluates were used for mass spectrometric analysis on an Orbitrap Velos instrument, fitted with a Thermo Easy-spray 50cm column.0-12h Tub-Gal4) or TM6BGFP . Embryos were collected on molasses agar plates and sorted into vials using lack of GFP fluorescence. A maximum of 50 larvae were sorted into each vial. Viability was assessed based on the number of pupated or eclosed individuals compared to the starting number of larvae in each vial.Males containing RNAi constructs were crossed to virgin females containing one of the Gal4 constructs balanced by CAG (https://www.ffmpeg.org/). The videos were then opened and converted to binary frames using Fiji/ImageJ. The wrMTrck plugin (http://www.phage.dk/plugins/wrmtrck.html) for ImageJ was used to assess the average speed of each larvae normalized to their body size (body lengths/second or BLPS).To assess larval locomotion, five wandering third instar larvae were set on a large molasses agar plate and placed in a recording chamber. Their crawling movements were recorded for at least 1 min on a digital camera (smartphone) at minimum zoom. Four recording were take for each set of larvae; at least 30 larvae total were recorded for each cross. The videos were transferred to a PC and converted to AVI files using ffmpeg (32P]ATP (PerkinElmer) using T4 PNK (NEB).Early third instar larvae (73-77 hr post egg-laying) were homogenized in TRIzol reagent (Invitrogen) and RNA was isolated according to the manufacturer’s protocol with the following modifications: a second chloroform extraction was performed, and RNA was precipitated with 0.1 volumes of sodium acetate and 2.5 volumes of ethanol rather than isopropanol. For Northern blotting, 2500 ng of total RNA was separated on Novex 10% TBE-Urea gels (Invitrogen). RNA was transferred to GeneScreen Plus Hybridization Transfer Membrane (PerkinElmer). Blots were dried, UV cross-linked, and pre-hybridized with Rapid-hyb Buffer . Probes were prepared by 5′-end labeling oligonucleotides with [γ-The oligonucleotide probe sequences are as follows:U1: 5′-GAATAATCGCAGAGGTCAACTCAGCCGAGGT-3′U2: 5′-TCCGTCTGATTCCAAAAATCAGTTTAACATTTGTTGTCCTCCAAT-3′U4: 5′-GGGGTATTGGTTAAAGTTTTCAACTAGCAATAATCGCACCTCAGTAG-3′U5: 5′-GACTCATTAGAGTGTTCCTCTCCACGGAAATCTTTAGTAAAAGGC-3′U6: 5′-CTTCTCTGTATCGTTCCAATTTTAGTATATGTTCTGCCGAAGCAAGA-3′U11: 5′-TCGTGATCGGAAACGTGCCAGGACG-3′U12: 5′-GCCTAGAAGCCAATACTGCCAAGCGATTAGCAAG-3′U4atac: 5′-AGCAATGTCCTCACTAGACGTTCATTGAACATTTCTGCT-3′U6atac: 5′-CCTAGCCGACCGTTTATGTGTTCCATCCTTGTCT-3′Following the PNK reaction, probes were purified using Microspin G-50 Columns . For hybridization, the blots were probed with the labeled oligonucleotides at 65°. The blots were then washed twice each in 2X SSC and 0.33X SSC at 60°. Blots were exposed to storage phosphor screens  and analyzed with an Amersham Typhoon 5 .To analyze knockdown efficiency, total RNA was treated with TURBO DNase (Invitrogen). Following a phenol/chloroform purification, 350 ng of RNA was converted to cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen). Primers for PCR are as follows:5S_F: 5′-GCCAACGACCATACCACGCTGAA-3′5S_R: 5′-AACAACACGCGGTGTTCCCAAGC -3′CG2941_F: 5′- TGTGGTATTGGCAGGACGGTCT-3′CG2941_R: 5′- CCTTGTGCTTCAATTTGCTCACTTGGTT -3′G4a-b-c_F: 5′- CCAGATAGCCTGCATGGAACATCG -3′G4a-b-c_R: 5′- CTCCCGCTTTAATGGATCATTGAGGG -3′https://doi.org/10.25387/g3.7473359.All fly strains, probe sequences and plasmids are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables. Supplemental material available at Figshare: Drosophila melanogaster proteins that might correspond to those known to be contained within the human SMN complex  of certain Gemin proteins in Drosophila .To identify  complex , we firsHezron/CG14164 encodes the Drosophila ortholog of Gemin6. Our bioinformatic analysis suggested that CG14164 is actually more closely related to human Lsm12, an Sm-like protein that contains a C-terminal methyltransferase domain  is slightly longer than Hezron/CG14164/Lsm12b (186aa) and that the two proteins begin to diverge only at their respective C-termini .-termini . There iCG31950/Sabbat was also identified by Gemin7 and member of the Drosophila SMN complex. We found that CG31950 was, in fact, more similar to an N-terminal acetyltransferase auxiliary subunit, Naa38 (D. hydei (XP_023177506.1), along with CG31950 from D. melanogaster (https://www.uniprot.org/uniprot/I3L310) diagnostic residues within the Naa38 orthologs are shared by CG31950. In contrast, the highly conserved regions of Gemin7, including the relative positions of insertions and deletions, do not track with CG31950  analysis (Bombus terrestris (Buff-tailed bumble bee) as the starting point for PSI-BLAST. We found that this seed sequence readily identified both vertebrate and invertebrate orthologs, along with three nearly identical D. melanogaster proteins, including CG2941, CG32783 and CG32786. An alignment of a subset of these identified proteins is presented in Fig. S1. As shown, the overall conservation of Gemin4 is rather modest. For comparison, the putative Gemin4 ortholog (GH21356) from a distantly related fruitfly, D. grimshawi, is also shown. Notably, an analysis of the predicted secondary structure of Gemin4 orthologs shows a high degree of conservation . Thus, despite the fact that the three D. melanogaster proteins  are most closely related to metazoan Gemin4, it is difficult to assign orthology on the basis of amino acid conservation alone.In contrast to Gem6-7-8, Gemin4 was originally thought to be lost from insects entirely ED6716  and virilis (D. virilis) groups each only have one CG2941-like gene and thus serve as outgroups for this analysis. Within the melanogaster group there appears to be ongoing genetic rearrangement of this genomic region, as certain species have up to five different copies of this gene, whereas others have only a single copy (D. melanogaster genes: Gemin4a (CG2941/Gaulos), CG32786) and Gemin4c (CG32783)Gemin4b , obtained from the Vienna Drosophila Resource Center, expresses a 339bp dsRNA that targets all three Gemin4 paralogs . Furthermore, the deficiency, Df(1)ED6716, used in the original genetic interaction screen also uncovers all three paralogs.The FlyAtlas anatomical and developmental expression database  or tubulin-Gal4 . Expression of either drivers or the responders alone had little to no effect on organismal viability (HMJ-22884) was essentially larval lethal , and roughly 20% of them eclose as adults. The phenotype of the Tub-Gal4 x HMJ-21933 cross was even more severe, as only ∼30% of the animals reached pupal stages and none progressed to adulthood , which takes into account the speed and size of each larva. For northern blotting, total RNA was harvested from early third instar (72-76hr) larvae, just prior to the beginning of the lethal phase of the RNAi. As shown in Drosophila Protein interaction Map) project showed that CG2941 co-precipitates with ectopically expressed, epitope-tagged SMN in S2 cells (https://interfly.med.harvard.edu). Subsequent quality control steps apparently removed CG2941 from the list of potential SMN interactors despite the fact that it also co-purifies with epitope tagged Gemin2 and Gemin3 . Third, we found that endogenous CG2941, CG32786 and CG32783 co-purify with SMN expressed from its native promoter in vivo in fly embryos , CG31950 (Sbat/Naa38) and CG2371 (Vlet/CommD10). An important question raised by our findings is whether or not the functions normally carried out by Hez, Sbat and Vlet are not orthologous to Gemin6, Gemin7 and Gemin8, respectively, it remains formally possible that they have been evolutionarily co-opted into the SMN complex in flies. Our inability to identify these three proteins as endogenous SMN binding partners by mass spectrometry  proteins can form homo- and hetero-dimers  is an auxiliary subunit of NatC  actually bind to the nascent peptide tunnel region of the ribosome in vivo is unclear. However, the fact that metazoan Naa38 is structurally similar to Sm proteins provides a plausible mechanism for their binding to the ribosome immediately following translation. Given that Gemin6 and Gemin7 are also members of the Sm-like superfamily of proteins  in Drosophila.First, Fischer and colleagues recently reported data suggesting that, following their translation, Sm proteins can remain bound to the ribosome near the exit tunnel, dissociating only after binding to the assembly chaperone pICln (bona fide member of the SMN complex (in flies or any other species), but it could potentially interact with SMN as part of its canonical function in N-terminal protein acetylation. More than 80% of human proteins are co-translationally modified on their N-termini  are very poorly conserved. Moreover, changing Ala2 to Gly is predicted to reduce the probability of N-terminal acetylation and recognition by the N-end-rule proteasomal degradation pathway  indicates that this protein is widely conserved. Moreover, genetic loss of function studies  derived from three different biological contexts might be co-opted into a novel Gemin subcomplex. A loss of the Gem6-7-8 subunit from the SMN complex in flies would suggest that either this subunit is not essential for basal metazoan viability or that other factors have compensated for deficiency of these proteins in


Bank1 (B-cell scaffold protein with ankyrin repeats 1) and Nfkb1 (nuclear factor kappa B subunit 1) were identified by additional QTL analysis. Expression of the Bank1 and Nfkb1 genes and their downstream target genes involved in the intracellular pathway  was investigated in mercury-exposed A.SW and B10.S mice by real-time PCR. Bank1 showed significantly lower gene expression in the A.SW strain after Hg-exposure, whereas the B10.S strain showed no significant difference. Nfkb1, Tlr9, Il6 and Tnf had significantly higher gene expression in the A.SW strain after Hg-exposure, while the B10.S strain showed no difference. This study supports the roles of Bank1 (produced mainly in B-cells) and Nfkb1 (produced in most immune cells) as key regulators of ANoA development in HgIA.Systemic autoimmune rheumatic disorders (SARD) represent important causes of morbidity and mortality in humans. The mechanisms triggering autoimmune responses are complex and involve a network of genetic factors. Mercury-induced autoimmunity (HgIA) in mice is an established model to study the mechanisms of the development of antinuclear antibodies (ANA), which is a hallmark in the diagnosis of SARD. A.SW mice with HgIA show a significantly higher titer of antinucleolar antibodies (ANoA) than the B10.S mice, although both share the same MHC class II (H-2). We applied a genome-wide association study (GWAS) to their Hg-exposed F2 offspring to investigate the non-MHC genes involved in the development of ANoA. Quantitative trait locus (QTL) analysis showed a peak logarithm of odds ratio (LOD) score of 3.05 on chromosome 3. Microsatellites were used for haplotyping, and fine mapping was conducted with next generation sequencing. The candidate genes  Failure to recognize self with non-self-antigens results in a disorder of the innate and adaptive immune systems, leading to the development of autoantibodies, but the details of this process are still unclear . Systemis have the highest susceptibility for developing ANoA, while H-2q and H-2f mice have intermediate susceptibility, and H-2a, H-2b, and H-2d mice are resistant to ANoA development [Different animal models are used to study SARDs. Mercury-induced autoimmunity (HgIA) in mice is an established and relevant model, which includes the development of antinucleolar antibodies (ANoA), immune complex (IC) deposits, hypergammaglobulinemia and polyclonal B-cell activation, and is controlled by multiple genes –16. One elopment .-/-, CD28-/-, and IFN-/- H-2s mice does not result in the development of ANoA [s show dissimilar severity of disease activity in HgIA. When comparing the two susceptible H-2s strains, A.SW and B10.S, the A.SW strain shows a more severe autoimmune manifestation by developing a higher serum ANoA titer, higher IgG IC titer, and higher serum IgG1 and IgG2a titers compared to the B10.S strain [However, knockout (KO) studies in mice have shown that non-H-2 genes also control the susceptibility to the development of systemic autoimmune disease –20. HgIA of ANoA , 20. AddS strain , 21–23.s (A.SW and B10.S) allowed us to investigate the non-H-2 genes, involved in the development of ANoA, by using GWAS. Mapping the quantitative trait loci (QTL) associated with an autoimmune trait was done with next generation sequencing (NGS), which allowed us to detect variants within the associated haplotype and identify the genes associated with the development of ANoA.Crossing two strains with the same H-2Bank1  and Nfkll types ) are potTo achieve DNA recombination in F2 mice, we crossed two susceptible strains (A.SW and B10.S) sharing the same H-2 haplotype. The phenotypic trait and DNA recombination in F2 offspring were used as a tool for GWAS. The phenotypic trait ANoA corresponds to staining of the nucleoli with clumpy nucleolar pattern, with 2–6 brightly staining dots in the nucleoplasm . The Hg-b) and one susceptible (H-2s) to developing ANoA, results in resistant F1 mice, suggesting a resistant dominant inheritance of the I-A region on H-2 [s loci, and the susceptibility for high ANoA titer is recessive.Logarithm of the odds (LOD) scores exceeding 3.82, determined by permutation testing, represent approximate thresholds for significant QTLs based on the normal distribution with a p-value of 0.05 . We founn on H-2 . We testNfkb1 gene (LOD 2.44) and two in the Bank1 gene (LOD 2.46 and 2.47)  were genotyped with 9 additional microsatellite markers spaced between 54.48–61.32 cM. We found a haplotype block between D3Mit247 (128 110 214 bp) and rs3676039 (136 217 610 bp) . We finend 2.47) .Bank1 between our two strains, A.SW and B10.S, by comparing them with the background strains (A and C57BL/6) using the Ensembl database [Bank1 is an adaptor/scaffolding protein expressed in most subpopulations of peripheral B-cells and has lower expression in plasmacytoid dendritic and myeloid cells but no expression in T-cells , 28. Ourdatabase . We foundatabase  a β-sheedatabase .Nfkb1 is also associated with ANoA development, and we found 5 SNPs  between background strains (A and C57BL/6), all with synonymous variants. There are different outcomes of silent mutations that, depending on their positions, may affect mRNA splicing, transcription and translation [Nfkb1 may have an impact on NF-kappaB gene/protein expression and function that may cause the ANoA titer differences.nslation . The SNPWe investigated the conservation of the SNPs in Bank1 at the nucleotide and amino acid levels see . Two of Bank1 and Nfkb1 genes are associated with the development of higher ANoA titer, we investigated the intracellular pathway by studying gene expression in spleen and believe that our findings mainly resemble what occurs in B-cells. We were interested to see if the gene expression of these proteins (NF-kappaB and Bank1) were affected by Hg-induced ANoA and found that the Bank1 gene expression was significantly lower in the A.SW strain (Bank1 upon Hg-exposure may lead to dysregulation of the B-cell receptor (BCR) downstream signaling pathway, which is regulated by Bank1 [Since Bank1 is a B-cell specific protein (no expression in T-cells) , 28 and W strain  on day 4W strain . The lowby Bank1 , 33.Nkfb1 gene expression was significantly higher after 12 days of Hg-exposure compared to day 0 and day 4 (p < 0.05) in the A.SW strain  on days 8 and 12 compared to day 0   . IL-6 re of ANoA . We beliBank1 DNA fragment (with exon 2), and the short Δ2 isoform (without exon 2)  , which iBank1 was significantly higher in the A.SW strain upon Hg-exposure   in the A.SW strain. The B10.S strain had significantly lower expression of the short Δ2 isoform compared to the full-length isoform after 4 and 12 days (p < 0.05). In humans, the full-length splice variant is associated with SLE, and this may be explained by the higher diversity in human genes and by the higher diversity between human populations compared to the two inbred mouse strains used in this study. The data for the susceptible B10.S strain are in concordance with what was found in human SLE patients, but we also found that higher expression of the short Δ2 isoform of Bank1 is associated with a higher ANoA titer.We analyzed the time-dependent expression of same splice variant and the differences in quantities between the two splice variants. The band intensity of the full-length variant in the A.SW strain showed no significant difference on all days when exposed to Hg . The B10 < 0.01) . The les < 0.01) . Our resBank1-deficient mice showed that Bank1 controls Tlr9 signaling via the p38-MNK1/2 pathway, in which Bank1-deficient showed higher Il6 expression [Tlr9. The A.SW strain showed significantly higher Tlr9 mRNA expression after 8 days (p = 0.043) compared to day 0 (Tlr9 gene expression after 4 days (p < 0.05) compared to day 0  are linked to ANoA, and Bank1 is highly expressed in B-cells but not in T-cells [A relevant question is, what activates the BCR to initiate the autoimmune manifestation in HgIA? Mercury has a high capacity of binding to thiol-containing proteins and affecting their structure . Fibrill T-cells , 28.However, both B- and T-cells are essential for development of ANoA in HgIA . We belis) strain is also a susceptible strain to HgIA, but it initiates a less severe autoimmune manifestation with the development of lower ANoA titer compared to the A.SW strain [The B10.S  and drinking water ad libitum. Studies were approved by the Laboratory Animal Ethics Committee, Linköping, Sweden, and all mice were treated humanely with regard to alleviating any suffering. Numbers of mice in each study are presented in 2/L (Fluka) in drinking water (n = 129), for 6 weeks before sacrifice. Blood was obtained from retro-orbital plexus after 6 weeks, and all animals were sacrificed by cervical dislocation, and tails, spleens and kidneys were obtained and stored at -70°C. Collected blood was allowed to clot for 2 hours before being centrifuged at 500 g for 15 minutes, after which the serum was removed and subsequently stored at -20°C.Female A.SW mice were obtained from Taconic, and male B10.S mice were obtained from the Jackson Laboratory. F1 hybrids were derived by crossing female A.SW mice and male B10.S mice. F2-hybrids  were obtained by crossing F1-hybrids. F2 mice (8–10 weeks old) received either tap water (n = 14) or 4.0 mg HgCl2/L (Fluka) in drinking water for 0, 4, 8 or 12 days. Each group consisted of 5–7 female mice. Mice were sacrificed by cervical dislocation, and spleens were sampled in RNAlater (Invitrogen) and stored at -70°C for subsequent analysis.Female A.SW mice were obtained from Taconic and female B10.S mice were obtained from the Jackson Laboratory. Mice (8–9 weeks old) were given 8.0 mg HgClSera from 129 Hg-exposed and 14 unexposed F2 mice were diluted 1:80–1:20,480 and incubated with HEp-2 cells  to detect binding of goat anti-mouse IgG antibodies  to cellular antigens . The ANoDNA isolated from tail, spleen or kidney from A.SW, B10.S, F1 and F2 mice was extracted using the Wizard SV Genomic DNA Purification System (Promega). These tissues were used to obtain the required amounts and concentrations of DNA. The quantity and purity of DNA was measured with a Qubit 3.0 fluorometer (Thermo Fisher Scientific).For the genome-wide genotyping, 129 Hg-exposed F2 samples were genotyped using the SNP&SEQ technology platform at Uppsala University. The Illumina Mouse Medium Density Linkage Panel contained 1449 single-nucleotide polymorphism (SNP) markers, of which 819 were polymorphic SNPs between the two strains A.SW and B10.S .Genotype-phenotype linkage analysis in F2 mice was performed to obtain genetic positions associated with ANoA. Quantitative trait loci (QTL) were identified based on the logarithm of odds (LOD) score profiles derived from a genome-wide single-QTL scan by Haley-Knott regression  with a hAdditional microsatellites were used to narrow down the region by haplotype analysis in which the QTL was found  in 129 HMus musculus USCS Mm10 [Thirty F2 mice, homozygous for the A.SW strain on associated haplotype, were selected for fine mapping. Fine mapping was based on next generation sequencing (NGS) of genes within the haplotype containing SNPs between background strains A (for A.SW) and C57BL/6 (for B10.S). SNPs were identified using the Ensembl  and MGI SCS Mm10 , using tSCS Mm10 . AlignedSCS Mm10 . VariantSCS Mm10 . GenotypTotal RNA was extracted from spleens of female A.SW and B10.S mice (n = 24) using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The quantity and purity of the RNA were measured using a Qubit 3.0 fluorometer (Thermo Fisher Scientific). RNA was diluted to 20 ng/μL and reverse-transcribed to cDNA by using the High-Capacity cDNA Archive kit (Applied Biosystems).Bank1, Nfkb1, Tlr9, Il6 and Tnf was measured with FAM (6-carboxyfluorescein) reporter dye–labeled probes . Target gene expression for d probes . The geot method .Bank1 from A.SW and B10.S mice, encompassing exon 2 and the upstream exon 1 and downstream exon 3 sequences, was amplified for splice variant detection in A.SW and B10.S mice . Fragments were amplified by 30 cycles of PCR under following conditions: denaturation at 94°C for 30 s, annealing at 60°C for 60 s, and extension at 72°C for 90 s. PCR products were separated on 1% agarose gel for 30 minutes at 120 Volts and measured with the GeneFlash Gel Documentation System (GeneFlash). Bands where quantified based on their relative intensities using ImageJ software 1.x [cDNA of ware 1.x .Prediction of the secondary structure of Bank1 protein was performed using the Chou & Fasman Secondary Structure Prediction (CFFSP) server. The cDNA sequences of background strains A.SW and B10.S were used to obtain the protein sequences that were used to predict the secondary structures of Bank1 by the Chou & Fasman algorithm . The cDNBank1 gene between background strains of 32 mammals Click here for additional data file.S2 TableSequencing was design for 11 genes targeting coding exons, and UTRs (5´UTR and 3´UTR).(DOCX)Click here for additional data file.S3 TableGene/protein names followed by gene symbols are presented.(DOCX)Click here for additional data file.S1 FigA) Missense variant rs30260564, F75L on exon 2 and B) missense variants rs50828248 and rs47442962, A375M on exon 7. Prediction of secondary structure was performed with the Chou & Fasman algorithm by the use of the online software server Chou & Fasman Secondary Structure Prediction (CFFSP) (Ashok Kumar T 2013). Query: amino acid position, Helix: α-helix structure, Sheet: β-sheet structure, Turns: structure folding.(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 FigFourteen mammalian species were selected by using Ensembl database for retrieval of amino acid sequences. Conserved region on amino acids were performed by aligning the multiple sequences with the use of Clustal X (version 2.1). rs50828248 and rs47442962 code for the same amino acid.(DOCX)Click here for additional data file.S4 FigDay 0 represents unexposed mice. Amplified cDNA encompass exon 2 and the upstream exon 1 and downstream exon 3 sequence. Full-Length represents cDNA with exon 2 and Delta 2 represents cDNA lacking exon 2.(DOCX)Click here for additional data file.S5 FigFor an SNP to be informative, it should vary between the genotypes of the A.SW and B10.S strains.(DOCX)Click here for additional data file.


The mitogen-activated protein kinase/extracellular signal-regulated (MAPK/ERK) pathway is a well-characterized signaling pathway during the development of various cancer types. ERK1 and ERK2, the two kinase effectors of MAPK cascade, exhibit high similarity. However, it is still unknown whether these two kinases are functionally different or in contrast functionally redundant during the development of breast cancer. We found that ERK1 expression levels were significantly lower in basal breast cancer compared with luminal breast cancer and normal breast tissues. RNA sequencing data suggested that ERK1 was associated with Hippo signaling pathway and cell proliferation in breast cancer cells. The gene set enrichment analysis (GSEA) further showed enrichment for YAP1 signaling pathway in breast cancer cell lines and tumors with low expression of ERK1. Silencing of ERK1 elevated YAP1 expression and TEAD activity in breast cancer cells. Additionally, ERK1 inhibited breast cancer cell proliferation via regulation of YAP1. The Kaplan-Meier analysis of data in patients with breast cancer suggested that, higher expression of ERK1 was associated with better prognosis, whereas, higher expression of ERK2 predicted poorer prognosis. These findings unveiled the role of ERK1 on regulation of YAP1 signaling pathway, indicating ERK1 as a negative regulator of breast cancer progression. Breast cancer was the leading cause of women death with more than 2,000,000 new cases and 600,000 deaths in 2018 [The four mitogen-activated protein kinase (MAPK) cascades have been widely studied, which are named according to MAPK tier component: the extracellular signal-related kinases (ERK1/2), Jun amino-terminal kinases JNK1/2/3), p-38 MAPK and ERK5 in mammalian cell , p-38 MA. NumerouThe Hippo signaling pathway, an evolutionarily conserved pathway, plays a crucial role in cell proliferation, apoptosis, differentiation, as well as cancer development . Yes-assRecent studies have reported that there is a crosstalk between Hippo-YAP pathway and MAPK-ERK pathway. Later, Wen Y et al found that the gene expression which took part in the phosphorylation of ERK1/2, was inhibited when YAP was silenced, and the protein levels of ERK and its downstream proteins were also reduced .in vitro and in vivo assays suggested that ERK1 inhibited breast cancer progression via downregulation of YAP1. Importantly, higher expression of ERK1 was correlated with better prognosis in patients with breast cancer and a predictor of better overall survival in patients receiving endocrine therapy. Higher expression of ERK2, in contrary, was associated with poorer prognosis.As for current study, we are the first to report the differences of distinct expression pattern, biological function and prognostic value between ERK1 and ERK2 in breast cancer. The Expression data for 50 breast cancer cell lines were downloaded from a previous study . The celTo study the global gene transcription regulated by ERK1, RNA sequencing (RNA-Seq) analysis was utilized to determine global gene expression change upon knockdown of ERK1 in T47D cells . ERK1 knTo investigate the association between ERK1 and YAP1 signaling, we analyzed expression of evolutionarily conserved signature of the YAP1 signaling genes described by Cordenonsi et al  with ERKTo validate the aforementioned observation, we silenced ERK1 in breast cancer cells. Western blotting showed that ERK1 knockdown increased the protein expression of YAP1 and phosphorylated YAP1 in luminal breast cancer cells, especially in MCF7 cells with higher ERK1 protein level , 4B. ERKin vitro experiments, in xenograft mouse models, silencing of ERK1 increased tumor volume and weight after injection of MCF7 cells, which was reversed by silencing of YAP1 . Samples were categorized by expression level that was above (high) or below (low) median ERK1 expression by quartiles (271 samples each group). The GSEA revealed a strong enrichment of YAP1 signaling-related genes in ERK1 low expression tumors compared with those with high ERK1 expression . FurtherYAP1 signaling was pivotal for cancer cell proliferation, metastasis and drug resistance . The KapIn 2006, Vantaggiato et al. discovered that transfection of ERK1 instead of ERK2 could inhibit transformation of NIH3T3 induced by stable expression of oncogenic Ras . Later, Previously, we discovered a crosstalk between the MAPK/ERK pathway and YAP1 signaling in cancer cells . Our finA previous report of mouse embryo discovered that the total activity of ERK1/2 was important for mouse development, which was in support of the notion that ERK1 and ERK2 showed functional redundancy . In contbona fide tumor suppressor role of ERK1 at least in breast cancer. High expression of ERK2, in contrast, was associated with poor prognosis in patients with breast cancer. It is known that basal breast cancer cells are particularly sensitive to RAF-MEK-ERK inhibitors compared with other subtypes, and ERK1 high expression predicted resistance to a MEK inhibitor [YAP1 was crucial for breast cancer cell proliferation, metastasis and drug resistance , the disnhibitor . Our finIn conclusion, our study demonstrated that ERK1 and ERK2 showed distinct expression pattern, prognostic value and function in regulation of YAP1 signaling in breast cancer. Our results supported a tumor suppressor role of ERK1 in breast cancer via suppression of YAP1 signaling pathway. Additional studies are needed to determine the molecular mechanism on regulation of YAP1 mRNA levels by ERK1; however, our data firstly suggested that ERK1 expression might be used as biomarker for prediction the prognosis in patients with breast cancer.2 at 37°C293T cells, breast cancer cell lines MCF7, T47D, MDA-MB-231, BT549 and HS578T were purchased from American Type Culture Collection . MDA-MB-435S, MDA-MB-231 and BT549 were cultured in DMEM , supplemented with 10% FBS . 293T, MCF7 and T47D were cultured in RPMI-1640 medium (Corning Cellgro) supplemented with 10% FBS (Hyclone). All cells were maintained in a humid incubator with 5% COERK1 siRNA pool (a pool of 3 target-specific siRNA) (sc-44205), ERK2 siRNA pool (a pool of 3 target-specific siRNA) (sc-44223) and control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology . YAP1 siRNA was bought from GenePharma . SiRNAs were transfected into MDA-MB-435S, MCF7, T47D, MDA-MB-231, HS578T and BT549 cells using Lipofectamine RNAi MAX  reagent according to manufacturer’s protocol. After 72 h, the cells were collected and subjected to the following experiments.Transcriptome libraries and RNA sequencing analysis were performed, aligned and normalized according to the Illumina Genome Analyzer II  manufacturer’s instruction. After clustering, the libraries were sequenced on an Illumina HiseqXTen platform using (2×150 bp) paired-end module. After initial quality control, the clean reads were mapped to the reference sequence by using TopHat2 software (v2.1.1.). The alignment files generated by TopHat2 were input to the Cufflinks software (v2.2.1), which was a program for the comparative assembly of transcripts and the estimation of their abundance in a transcriptome sequencing experiment by using the measurement unit fragments per kilobase of transcript per million mapped reads (FPKM). After using Cuffmerge program to merge transcripts of each sample in different materials and stages into a single gtf file that was used to identify differentially expressed genes, Cuffdiff program was used to find differentially expressed genes (DEGs). The differentially expressed genes were identified with p value ≤0.05 and a fold-change of ≥2. Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to analyzed the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway modulated by the knockdown of ERK1 or ERK2 .Total RNA of MDA-MB-435S, MCF7, T47D, HS578T and BT549 cells were extracted using TRIzol reagent (Invitrogen). The concentration and quality of RNA were measured with NanoDrop2000 . The RNA was reverse transcribed into first-strand cDNA with PrimeScript RT reagent kit . RT-qPCR was performed with SYBR Premix Ex Taq (TaKaRa) on a CFX96 Real-Time PCR System . The primer sequences were listed in Antibodies for ERK1/2 , phospho-ERK1/2 (Thr202/Tyr204) , LATS1, LATS2, p-LATS1/2 (Thr1079)  and YAP1  were purchased from cell signaling technology . GAPDH mouse monoclonal antibody  was obtained from Kangchen . Secondary HRP-conjugated antibodies against mouse  and rabbit  were products of ProteinTech . Protein lysates were prepared with RIPA lysis buffer . The concentration of protein lysates was determined with a BCA Protein Assay Kit . For western blotting, lysates with 20 μg protein per lane were loaded on an 8% SDS-PAGE gel. The proteins were separated and transferred to a PVDF membrane. The membrane was then blocked in 5% non-fat milk at room temperature for 1 h. After that, the membrane was incubated with primary antibodies overnight at 4°C On the next day, the membrane was incubated with indicated secondary antibody at room temperature for 1 h. The blots were developed with SuperSignal West Femto Maximum Sensitivity Substrate .T47D cells were grown on glass slide in a 24-well plate. After transfection of control siRNA or ERK1 siRNA and incubation for 72 h, the immunofluorescence was performed as we published before . The nucFor stable knockdown of ERK1, control scramble shRNA and ERK1 shRNA (1-3 sequences) were cloned into pLKO.1 plasmid. The sequences were: control shRNA: 5′-CCGGTAAGGCTATGAAGAGATACCTCGAGGTATCTCTTCATAGCCTTATTTTTG-3′, ERK1 shRNA1: 5′-CCGGTCATCGGCATCCGAGACATTCCTCGAGGAATGTCTCGGATGCCGATGATTTTTG-3′, ERK1 shRNA2: 5′-CCGGGACAGACATCTCTGCACCCTGCTCGAGCAGGGTGCAGAGATGTCTGTCTTTTTG-3′, ERK1 shRNA3: 5′-CCGGCAACATGAAGGCCCGAAACTACTCGAGTAGTTTCGGGCCTTCATGTTGTTTTTG-3′. 293T cells were transfected with the lentivirus vector and packaging plasmids. At 48 h after transfection, viruses were collected and replaced with fresh medium. At 12 h after incubation, the viruses were collected again and used for infection of T47D cells. Stably infected T47D cells were selected with 2 μg/mL puromycin  for 3 days.The activity of TEAD transcription factor was detected with the luciferase reporter assay. The 8×GTIIC-luciferase plasmid  and Renilla luciferase pRL-TK plasmid  were co-transfected into T47D and MCF7 cells with Lipofectamine 3000 (Invitrogen) following manufacturer’s protocol. After 48 h, cells were harvested, the firefly and renilla luciferase activities were measured by using the Dual-Luciferase Reporter Assay Kit (Promega) according to the manufacturer's instructions. The firefly luciferase was normalized to Renilla luciferase, and the relative firefly activity of ERK1 siRNA group was compared to control group to manifest the relative transcription activity of TEAD.The cell viability was determined using a CCK-8 kit  following manufacturer’s protocol. Briefly, 10 μL CCK-8 solution was added into indicated well and sustained for 2 h. After that, the medium was transferred into wells of a new 96-well plate. The absorbance at 450 nM was detected to manifest the cell viability.The cycloheximide (CHX) chase assay was used to analyze the stability of protein. CHX was purchased from MedChemExpress . Cells were firstly transfected with ERK1 siRNA, ERK2 siRNA or control siRNA for 72 h, CHX (50 μg/ml) was then added into medium for indicated hours. The YAP1 protein levels were detected by Western blotting.http://www.broadinstitute.org/gsea/msigdb/genesets.jsp?collection=C6, Broad Institute, MSigDB, Version 4.0) were used for the analysis with default settings and 1,000 gene set permutations. One gene set was filtered out by its size (<15 or >500 genes) and excluded from the analysis. Correlation between gene mRNA expression in the gene set of cell lines or human breast tumors were analyzed by calculating the Pearson correlation coefficient. Heat maps were generated using Morpheus software (https://software.broadinstitute.org/morpheus).The gene expression profile of 50 breast cancer cell lines were retrieved from a previously published work . Cell liFor analysis of ERK1, ERK2 and YAP1 expression in breast cancer cell lines, we retrieved mRNA expression data of 50 breast cancer cell lines from a previously published work . In addiTo evaluate the expression pattern of ERK1 and ERK2, the human breast tumor microarray data set was obtained from the NCBI-GEO database (GSE18229-GPL1390) containing 16 normal breast samples and 180 tumor samples with subtype classification according to Prat et al .http://kmplot.com/analysis/index.php?p=service&cancer=breast) was used to explore the association between ERK1/2 expression and breast cancer patient outcome. Cases were divided by expression which was above (high) or below (low) median ERK1 (212046_x_at) or ERK2 (212271_at) expression.To assess the prognostic value of ERK1 and ERK2, Kaplan-Meier Plotter (KM plotter) database  (http://7) were transfected with indicated shRNAs , counted, suspended in PBS, and mixed with Matrigel . Female athymic STOCK-Foxn1nu/Nju mice were purchased from Model Animal Research Center of Nanjing University. Mice were randomly separated into 3 groups (n=4) and the PBS–Matrigel mixture were injected into the mammary fat pads. A 0.72-mg E2 60-day release pellet  was implanted subcutaneously on the dorsal side of each mouse a day before injection of tumor cells. Tumor volume was measured every week from the time when tumor was palpable (2 weeks later) until the mice were sacrificed (week 6). Tumor volumes were calculated by measuring width and length of tumors with a Vernier caliper and calculated by formula as listed: Volume = (Length × Width × Width)/2. All animal studies were performed in accordance with the guidelines for the care and use of laboratory animals of Nanjing Normal University.MCF-7 cells (1X10All data were analyzed with Graphpad Prism 6.0 and presented as mean ± SD. The differences between two groups were analyzed using Student’s t test. The differences among three groups were analyzed using one-way ANOVA followed by Newman Keuls analysis. A p value less than 0.05 was considered statistically significant. All experiments were repeated at least 3 times.Supplementary FiguresSupplementary Table 1