RAD51 and RAD52 using the Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant from Tolerant (SIFT) algorithms and observed the effect of mutations in highly conserved domains of RAD51 and RAD52 on DNA damage repair in a Saccharomyces cerevisiae-based system. We identified a number of rad51 and rad52 alleles that exhibited severe DNA repair defects. The functionally inactive SNPs were located near ATPase active site of Rad51 and the DNA binding domain of Rad52. The rad51-F317I, rad52-R52W, and rad52-G107C mutations conferred hypersensitivity to methyl methane sulfonate (MMS)-induced DNA damage and were defective in HR-mediated DSB repair. Our study provides a new approach for detecting functional and loss-of-function genetic polymorphisms and for identifying causal variants in human DNA repair genes that contribute to the initiation or progression of cancer.Systems for the repair of DNA double-strand breaks (DSBs) are necessary to maintain genome integrity and normal functionality of cells in all organisms. Homologous recombination (HR) plays an important role in repairing accidental and programmed DSBs in mitotic and meiotic cells, respectively. Failure to repair these DSBs causes genome instability and can induce tumorigenesis. Rad51 and Rad52 are two key proteins in homologous pairing and strand exchange during DSB-induced HR; both are highly conserved in eukaryotes. In this study, we analyzed pathogenic single nucleotide polymorphisms (SNPs) in human  Genomic instability due to defects with DNA repair proteins causes various inherited genetic disorders in humans and also leads a generalized cancer predisposition. DNA damage, including double-strand breaks (DSBs) and cross-linkages, occurs spontaneously during the normal cell cycle and upon exposure to ionizing radiation or mutagenic chemicals , 2. DefeRad51 and Rad52 are key proteins involved in DSB repair by HR and are conserved between humans and yeast –9. Rad51Many human diseases have a strong genetic component, and human genetic studies have successfully determined the causes of numerous rare Mendelian disorders . MissensRad51 and Rad52 to determine if these missense mutations also result in loss-of-function or lethality in Saccharomyces cerevisiae. Specifically, we analyzed the JMs and recombination products of rad51 and rad52 mutants with causal variants that are associated with deficient strand exchange activity of the HR pathway. We validated the findings for deleterious mutations associated with human RAD51 and RAD52 and assessed their possible complementary roles as DNA damage repair proteins using a model system.Although many mutations in DNA repair genes have been reported, it is difficult to determine their clinical significance. Here, we describe functional analysis of causal variants in human RAD51 (8 SNPs) and RAD52 (4 SNPs) from the NCBI SNP database (http://genetics.bwh.harvard.edu/pph) and SIFT (http://sift.jcvi.org) programs were used to predict the effects of amino acid substitutions on protein function and structure, respectively  within database . We compectively . From thnditions . Specifinditions .S. cerevisiae. We aligned the Rad51 and Rad52 sequences in various species, including Homo sapiens and S. cerevisiae, Pan troglodytes, Canislupus, Bos taurus, Rattus norvegicus, Mus musculus, Gallus gallus, Danio rerio, Kluyveromyces lactis, and Eremothecium gossypii using the ClustalW program for multiple sequence alignment of proteins , which were selected using the NCBI SNP database, were located at the DNA binding site in both human and S. cerevisiae Rad52 proteins  HIS4::LEU2 and paternal (“Dad”) his4x::LEU2 loci, interhomolog JMs (IH-JMs), representing interhomolog interactions, could be distinguished from intersister JMs (IS-JMs), representing “Mom + Mom” interactions. We could identify the transient formation of IH-JMs and IS-JMs, in addition to replication Y-arc signals, in the 2D-gel analysis  and two-dimensional (2D) gel electrophoresis of the U2 locus  40]. Ye. YeHIS4LU2 locus . I-SceI eviously . XhoI-reantified . DSBs wigle band . Furthergle band . JMs froanalysis .rad51 and rad52 alleles in DSB repair, we prepared a galactose inducible I-SceI system as described above. In the WT strain, DSBs were first detected 30 min after the addition of galactose to the YP medium; they peaked at 1h, and were processed efficiently to produce recombinants  mechanism, which yields non-crossovers or disappears by DSB-end hyperresection.The structure of mitotic recombination intermediates was further investigated with native/native 2D gel analysis in which the DNA fragments were separated based on molecular weight in the first dimension and shape in the second dimension. In all experiments, cellular DNA was subjected to psoralen-UV crosslinking prior to DNA isolation immediately after harvesting cells from each time point. Psoralen-UV crosslinking stabilizes recombination-associated JMs such as single-end invasions (SEIs) and double-Holliday junctions (double-HJs). After DNA replication, “Mom + Mom” IS-JM species, representing sister chromatid interactions established for the repair of DSBs, were expected to form a JM of 11.8 kb . “Mom + les Figs  and S6. rad51 and rad52 alleles affect the efficiency of spore formation. The rad51 and rad52 alleles were cultured to induce sporulation in SPM media for 24 h at 30°C, and spore viability was analyzed after tetrad dissection. In WT cell, 94% of tetrads underwent normal sporulation based on the levels of viable spores, however, the spores of rad51Δ, rad51-F317I, rad52-R52W, and rad52-G107C cells were not viable, in accordance with the physical DNA analysis results  inactivated the DSBR pathway and prevented formation of viable spores.During meiosis, yeast cells with unsegregated DNA fail to form mature spores. We investigated whether deleterious  results . The radectively . This reRad54B, has been identified in human cells and encodes a protein that consists of 747 amino acids and is 52% identical to yeast Rad54 [rad54 alleles as causal variants because the sequence similarity of the functional domain was low between human and yeast.HR is a fundamental mechanism for the repair of DNA DSBs caused by ionizing radiation, ultraviolet light, chemical agents, and replication errors. Mitotic HR supports genomic stability and suppresses tumorigenesis via the proper repair of DSBs . Mitoticst Rad54 , 49. HowS. cerevisiae alleles to examine mammalian genes have been well established and successfully used for screening mutants affecting DNA damage repair. In this study, the approach included bioinformatic and structure-based predictions were used to identify missense mutations in human RAD51 and RAD52 genes. We identified a number of deleterious alleles of the human Rad51 and Rad52 proteins that were defective in the strand exchange activity of HR using a yeast-based system. Based on the in vivo analysis, we estimated that the accuracy of functional variation prediction by SIFT and PolyPhen was 60%, which is impressive compared to previous estimates [rad51-F317I, rad52-R52W, and rad52-G107C mutants, similar to rad51 or rad52 deletion mutants. These results suggested that the ATPase activity domain of Rad51 and the DNA-binding domain of Rad52 are crucial for DNA repair and that mutations in these regions might be associated with various cancers.Systematic approaches using stimates , 51. FurModel systems for the analysis of DNA damage repair have the potential to be used for identifying and understanding novel genes and pathways relevant to human cancer. Further studies in human patients with various cancer types may support these new insights into potential disease mechanisms and therapeutic strategies. We anticipate that the deleterious mutations of human HR genes verified herein could contribute to genomic instability and be related to cancer initiation and progression.RAD51 and RAD52 were selected from the National Center for Biotechnology Information Single Nucleotide Polymorphism (NCBI SNP) database using the following methods. (i) Human RAD51 and RAD52 gene sequences were compared to corresponding sequences in other species, including yeast. (ii) SNPs in the functional domain were selected. (iii) The selected human and yeast SNPs were matched by position. (iv) Finally, the impact of the amino acid substitutions was predicted using Sorting Intolerant From Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen) to select “damaging” and “deleterious” SNPs. The open reading frames (ORFs) of RAD51 and RAD52 were aligned by EBI ClustalW . SNPs were selected from the NCBI SNP database (http://www.ncbi.nlm.nih.gov/snp/) and confirmed to be located in exons. The RAD51 and RAD52 SNPs were processed using SIFT and PolyPhen. SNPs located in exons were examined using SIFT and PolyPhen (http://asia.ensembl.org/Homo_sapiens/Gene/) to select deleterious and damaging SNPs.Single nucleotide polymorphisms (SNPs) in HIS4LEU2 locus and I-SceI cleavage site have been described previously [SK1 strains were used in this study and are described in eviously .RAD51 and RAD52 open reading frames (ORFs) were amplified by polymerase chain reaction (PCR) using primers flanking the ORFs and containing restriction sites to produce HindIII and PacI sites. PCR products were digested with HindIII and PacI and cloned into the corresponding sites of pFA6a-NT1 plasmid vectors. To create rad51 and rad52 mutations, the ORFs and selection marker regions were amplified using the pairs of primers (shown in RAD51 primers were generated as follows (mutant bases are underlined): rad51-F317I forward, 5'-CTTTGCAAAGGCTGGCCGACCAAATT-3'; rad51-K371Q forward, 5'-GGTAAGGGATGTCAAAGATTATGCCAA-3'; and reverse primer, 5'-GAAAAAGAGGAGAATTGAAAGTAAACCTGTGTAAATAAATAGAGACAAGAGACCAAATAC GTTAAAGCCTTCGAGCGACCC-3'. RAD52 primers were generated as follows (mutant bases are underlined): rad52-G41R forward, 5'-ATATCTCCAAGAGAGTTGGGTTTAGA-3'; rad52-R52W forward, 5'-GATTGCATACATCGAAGGTTGGTGG-3'; rad52-G107C forward, 5'-CGGGACTTATAGGGAAGATATTTGT-3'; and reverse primer, 5'-AGGATTTTGGAGTAATAAATAATGATGCAAATTTTTTATTTGTTTCGGCCAGGAAGCGTTGTTAAAGCCTTCGAGCGTCCCAAAA-3'. PCR products were transformed into the SK1 strain and the cells were then grown on hygromycin B (200 mg/L) plates for 3 days at 30°C. The RAD51 and RAD52 ORFs from single colonies grown on hygromycin B plates were confirmed by DNA sequencing using the ABI Prism BioDye Terminator version 3.1.The shown in . ForwardSceI expression. After 45 min, 3% glucose was added to repress I-SceI expression. Cells were harvested at the time points.Diploid cells were cultured in YPD  for 24 hr. To synchronize cells in the G1 phase, the YPD cultures were diluted 1:500 and grown in SPS  for 18 hr at 30°C. Synchronized cells were then washed once and resuspended with fresh YP including 2% galactose to induce I-XhoI and precipitated in > 99% ethanol. Digested DNA samples were allowed to separate in 0.6% agarose gel in TBE buffer  without ethidium bromide at 2 V/cm for 24 hr. Gels were stained with ethidium bromide, and the lanes were cut out and placed in a 2D gel tray. Electrophoresis in the second dimension was carried out at 5 V/cm for 6 hr. Gels were subjected to Southern hybridization analysis using Probe A , and spotted onto YPD plates containing methyl methane sulfonate . After incubation at 30°C for 3 days, the plating efficiency and cell growth were evaluated.Cells were grown to saturation in YPD at 30°C overnight and were serially diluted by 10-fold decreases  or anti-Rad52 (Cat. No. sc-50445) polyclonal antibody were used for immunostaing. The following day, the cells were washed five times for 5 min each time with 2% BSA in PBS, then incubated with FITC-conjugated anti-rabbit IgG (H+L) secondary antibody  at a dilution of 1:200 for 1 h at room temperature. Cells were washed three times with blocking buffer, and then rinsed with PBS at room temperature. The nuclei were incubated with DAPI and mounted using Dako fluorescent mounting medium . Immunofluorescence was visualized on an Olympus BX53 Fluorescence Microscopy System .http://swissmodel.expasy.org/) and visualized by PyMOL . Overall structural changes induced by side chain variations could be measured by comparisons of the root-mean-square deviation (RMSD) of C-alpha. The overall RMSD reflects the average distance between all of the backbone atoms of wild type (WT) and variant proteins. The RMSDs of Calphas of consecutive 10-amino acid-long fragments (RMSD10aa) were measured using PyMOL.Structural alterations of Rad51 and Rad52 at the protein level induced by SNPs were determined by SWISS-MODEL Click here for additional data file.S2 FigH. sapiens and S. cerevisiae, P. troglodytes, C. lupus, B. taurus, R. norvegicus, M. musculus, G. gallus, D. rerio, K. lactis, E. gossypii by use of clastalW program. Red box, selected SNPs for this study.Rad51 and Rad52 sequences are aligned in various species, including (TIF)Click here for additional data file.S3 Figrad51-F317, rasd51-K371Q, rad52-G41R, rad52-R52W, and rad52-G107 cells were stained with rabbit anti-Rad51 or anti-Rad52 polyclonal antibody, followed by staining with anti-IgG conjugated with FITC. DNA was stained with DAPI.Cells were incubated with 0.1% MMS and subjected to immunofluorescence as described in Materials and Methods. WT, (TIF)Click here for additional data file.S4 FigSensitivity to DNA damage was induced by methyl methane sulfonate (MMS). Cells were cultured in YPD liquid for 24 h, and then spotted onto YPD plates containing 0.02% MMS.(TIF)Click here for additional data file.S5 FigWT (KKY940), rad51Δ (KKY1089), rad51-F317I (KKY1086), rad51-K371Q (KKY1091), rad52-G41R (KKY1143), rad52-R52W (KKY1145), and rad52-G107C (KKY655). (B and D) Quantification of DSBs and recombinants.(A and C) One-dimensional gel analysis of DSB and recombinants formation over the time course in (TIF)Click here for additional data file.S6 Figrad51Δ (KKY1088), rad51-F317I (KKY1086), rad51-K371Q (KKY1091), rad52Δ (KKY1142), rad52-G41R (KKY1143), rad52-R52W (KKY1145), rad52-G107C (KKY655). (B) Quantification of JM formation.(A) Two-dimensional gel of JM formation over the time course in WT (KKY940), (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


D that is only 5.6-fold weaker than for RNA:DNA duplexes. Most IP protocols do not pre-clear the genomic nucleic acid with RNase A to remove free RNA. Fold back of ssRNA can readily generate RNA:RNA duplexes that may bind the S9.6 antibody, and adventitious binding of RNA may also create short RNA:DNA regions. Here we investigate whether RNase A is needed to obtain reliable IP with S9.6.Long genomic R-loops in eukaryotes were first described at the immunoglobulin heavy chain locus switch regions using bisulfite sequencing and functional studies. A mouse monoclonal antibody called S9.6 has been used for immunoprecipitation (IP) to identify R-loops, based on the assumption that it is specific for RNA:DNA over other nucleic acid duplexes. However, recent work has demonstrated that a variable domain of S9.6 binds AU-rich RNA:RNA duplexes with a KAs our test locus, we chose the most well-documented site for kilobase-long mammalian genomic R-loops, the immunoglobulin heavy chain locus (IgH) class switch regions. The R-loops at this locus can be induced by using cytokines to stimulate transcription from germline transcript promoters. We tested IP using S9.6 with and without various RNase treatments. The RNase treatments included RNase H to destroy the RNA in an RNA:DNA duplex and RNase A to destroy single-stranded (ss) RNA to prevent it from binding S9.6 directly (as duplex RNA) and to prevent the ssRNA from annealing to the genome, resulting in adventitious RNA:DNA hybrids. We find that optimal detection of RNA:DNA duplexes requires removal of ssRNA using RNase A. Without RNase A treatment, known regions of R-loop formation containing RNA:DNA duplexes can not be reliably detected. With RNase A treatment, a signal can be detected over background, but only within a limited 2 or 3-fold range, even with a stable kilobase-long genomic R-loop.Any use of the S9.6 antibody must be preceded by RNase A treatment to remove free ssRNA that may compete for the S9.6 binding by forming RNA:RNA regions or short, transient RNA:DNA duplexes. Caution should be used when interpreting S9.6 data, and confirmation by independent structural and functional methods is essential.The online version of this article (doi:10.1186/s13104-015-1092-1) contains supplementary material, which is available to authorized users. Recent work has shown that a single-chain variable domain of the S9.6 antibody can bind RNA:RNA duplexes with an affinity that is only 5.6-fold weaker than to RNA:DNA duplexes, raising the serious concern that S9.6 can indeed cross react with RNA species [While S9.6 antibody can recognize RNA:DNA duplexes -28, comp species . Most of species -28,30,31 species , which i species -35.6 cells/ml were supplemented with anti-CD40 (eBioscience Cat. No. 16-0404-86), IL-4 (R&D Cat. No. 404-ML-010) and TGF- β1 (R&D Cat. No. 240-B-002) for 24 hours.CH12F3.2a and its derivative cells were cultured in RPMI medium supplemented with 10 % FCS and 50 μM β-mercaptoethanol . For expATCC HB-8730 hybridoma line (generously provided by Bradley Cairns) was cultured in a CELLine 1000 bioreactor  according to manufacturer’s instructions. Harvested antibody (culture supernatant) was purified on a column packed with Protein G Sepharose 4 Fast Flow  equilibrated with 1x phosphate buffered saline (PBS).Genomic DNA from CH12F3.2a cells with and without cytokine stimulation was prepared by overnight proteinase K digestion, phenol-chloroform extraction and ethanol precipitation. Genomic DNA was digested with EcoRI; importantly, RNase A was added at this step to prevent S9.6 antibody binding to RNA species in subsequent steps in the experiments with RNase A treatment . Five mWe stimulated CH12F3.2a cells (which are capable of robust cytokine-dependent CSR in culture) for 24 hr before genomic DNA was harvested and digested in preparation for immunoprecipitation (IP) as described previously . The traIn these IP experiments , we findIt is surprising that we detected signals clearly above the background within the fragments far upstream and downstream of the alpha locus even though these signals do not change with cytokine stimulation. We have previously documented that the region upstream of the germline transcript promoter for the IgH alpha locus and the downstream region do not harbor any R-loops, regardless of cytokine treatment ,3,4,33. We also examined the beta-actin gene in the DNA samples from the same IP experiments. The beta-actin gene is at another location in the genome, and it has not been documented to have any extended R-loop by physical methods. The signal at the beta-actin gene is similar to all regions other than IgH Sα Figure , Group 4Taken together, the S9.6 antibody appears to have good specificity for binding to RNA:DNA hybrids, but its ability to distinguish extended R-loops versus small RNA:DNA hybrids arising during DNA replication (RNA primer sites) or from other causes is highly uncertain. With the potential high background due to the antibody binding to non-R-loop RNA:DNA hybrids, the dynamic range of the R-loop signal is greatly affected.In all the IP experiments of DNA samples without RNase A treatment regardless of cytokine stimulation, qPCR did not detect any signal above background in any regions examined (Figure The IgH class switch region R-loops can be up to kilobases in length, and thus provide the largest possible target upon which to test the S9.6 antibody for detection of RNA:DNA duplex in the mammalian genome ,2. DespiWe will immediately provide anyone with our primary data.


The Ras-GAP SH3 domain–binding proteins (G3BP) are essential regulators of the formation of stress granules (SG), cytosolic aggregates of proteins and RNA that are induced upon cellular stress, such as virus infection. Many viruses, including Semliki Forest virus (SFV), block SG induction by targeting G3BP. In this work, we demonstrate that the G3BP-binding motif of SFV nsP3 consists of two FGDF motifs, in which both phenylalanine and the glycine residue are essential for binding. In addition, we show that binding of the cellular G3BP-binding partner USP10 is also mediated by an FGDF motif. Overexpression of wt USP10, but not a mutant lacking the FGDF-motif, blocks SG assembly. Further, we identified FGDF-mediated G3BP binding site in herpes simplex virus (HSV) protein ICP8, and show that ICP8 binding to G3BP also inhibits SG formation, which is a novel function of HSV ICP8. We present a model of the three-dimensional structure of G3BP bound to an FGDF-containing peptide, likely representing a binding mode shared by many proteins to target G3BP. Stress granules (SGs) are dynamic aggregates of proteins and translationally silenced mRNA that are formed in cells upon various stress conditions, such as virus infection. SGs are thought to be antiviral, and many viruses have hence evolved countermeasures to prevent their formation, often targeting the essential SG protein G3BP. Here, we show that several otherwise unrelated viral and cellular proteins all bind G3BP with the sequence motif FGDF, and thereby repress SG formation: the non-structural protein 3 (nsP3) of the Old World alphavirus Semliki Forest virus ; the protein ICP8 of herpes simplex virus; and in addition, the cellular protein USP10 (an SG component and protein deubiquitinase that stabilises e.g. the tumor suppressor p53). In this work, we also present and validate a model of the three-dimensional structure of G3BP bound to an FGDF-containing peptide. The FGDF-mediated G3BP binding represents an attractive target for therapeutic interventions against a range of diverse viral infections, and may also regulate the p53-stabilising function of USP10 in cancers. The Ras-GAP SH3 domain–binding proteins (G3BP) are multifunctional RNA-binding proteins, present in two forms, G3BP-1 and G3BP-2 (here collectively referred to as G3BP). They have a well-described importance in mediating the formation of RNA stress granules (SG), both in cells exposed to environmental stress and viral infections . The assSGs are induced by many virus infections and in turn, viruses have evolved many countermeasures, often targeting G3BP . SG asseIn this work, we set out to precisely define the characteristics of the G3BP-binding motif in the Old World alphavirus nsP3. We found that the core binding motif consists of two phenylalanine residues separated by a glycine and an aspartate residue (FGDF). Mutation of either of these residues prevented binding to either G3BP-1 or G3BP-2. We also find FGDF motifs at the N-terminus of USP10 and at the C-terminus of herpes simplex virus (HSV)-1 protein ICP8, and confirmed that these motifs similarly bind G3BP and block SG induction. Finally, we generated a molecular model of the G3BP/FGDF peptide interaction and validated the model by site-directed mutagenesis. These results reveal the mechanism by which at least two pathogenic viruses target G3BP. Moreover, they also provide a molecular basis for the regulation of USP10 activity by assembly of the inhibitory USP10/G3BP complex.The C-terminal L/ITFGDFD repeat motifs, which constitute the G3BP-binding site of nsP3, are well conserved in the Old World alphaviruses  with parFGDFD motifs, hereafter referred to as SFV-F3A). Previously, we showed that SFV-Δ789, lacking nsP3 residues 449–472, including the G3BP-binding sites displayed slower processing of the P34 precursor -F-[DE], F-G-[DES]-F-x-[DE], F-G-[DES]-F-x-x-[DE], F-G-[DES]-F-x-x-x[DE], F-G-[DES]-F-x-x-x-x-[DE]. Table is grouped according to FGDF, FGEF and FGSF motifs and within the groups sorted according to gene name.(PDF)Click here for additional data file.S2 TableThe protein sequence database UniProtKB, filtered on the taxonomy viruses, was scanned for the following motifs F-G-[DES]-F-[DE], F-G-[DES]-F-x-[DE], F-G-[DES]-F-x-x-[DE], F-G-[DES]-F-x-x-x[DE], F-G-[DES]-F-x-x-x-x-[DE].Table is grouped according to FGDF, FGEF and FGSF motifs and within the groups sorted according to gene name. The FGxF motif in the Togaviridae family can be found in the non-structural protein 3 (nsP3).(PDF)Click here for additional data file.S3 TableRestriction sites are underlined. Overlapping sequences are shown in italic and nucleotides encoding amino acid substitutions are shown in bold.(PDF)Click here for additional data file.S4 TableAntigen, species, application and source of all antibodies used in the study.(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Fifty-five percent of individuals with HLA-B*57:01 exposed to the antiretroviral drug abacavir develop a hypersensitivity reaction (HSR) that has been attributed to naïve T-cell responses to neo-antigen generated by the drug. Immunologically confirmed abacavir HSR can manifest clinically in less than 48 hours following first exposure suggesting that, at least in some cases, abacavir HSR is due to re-stimulation of a pre-existing memory T-cell population rather than priming of a high frequency naïve T-cell population.To determine whether a pre-existing abacavir reactive memory T-cell population contributes to early abacavir HSR symptoms, we studied the abacavir specific naïve or memory T-cell response using HLA-B*57:01 positive HSR patients or healthy controls using ELISpot assay, intra-cellular cytokine staining and tetramer labelling.in vitro in one hundred percent of abacavir unexposed HLA-B*57:01 positive healthy donors. Abacavir-specific CD8+ T cells from such donors can be expanded from sorted memory, and sorted naïve, CD8+ T cells without need for autologous CD4+ T cells.Abacavir reactive CD8+ T-cell responses were detected We propose that these pre-existing abacavir-reactive memory CD8+ T-cell responses must have been primed by earlier exposure to another foreign antigen and that these T cells cross-react with an abacavir-HLA-B*57:01-endogenous peptide ligand complex, in keeping with the model of heterologous immunity proposed in transplant rejection. Abacavir hypersensitivity reaction (HSR) is a potentially life threatening CD8+ T cell mediated, HLA-B*57:01 restricted syndrome previously occurring in 5–8% of those treated with the drug, but now prevented by HLA-B*57:01 screening prior to abacavir prescription . Abacaviin vitro findings are therefore compatible with the 100% negative predictive value of the test but not the 55% positive predictive value. Furthermore, the onset of abacavir HSR symptoms can occur as early as 36 hours after first exposure, characteristic of re-activation of pre-existing memory T cells but also as late as 3 weeks, which is more characteristic of either a delayed expansion of pre-existing memory CD8+ T cells or with the in vivo expansion of naïve CD8+ T-cell responses. Here we report findings that support the contribution of both mechanisms; we detect abacavir responsive CD8+ T cells ex vivo within PBMC from HLA-B*57:01 positive abacavir-unexposed donors and also demonstrate that abacavir can drive the expansion of CD8+ T-cell responses from both sorted naïve or memory T cells from HLA-B*57:01 positive donors. We therefore propose a model in which an HLA-B*57:01 restricted CD8+ memory T-cell response to a currently unknown pathogen specific epitope cross-recognizes an endogenous peptide that is only presented by HLA-B*57:01 in the presence of pharmacological levels of abacavir. Exploiting the fact that vaccination and immunity to yellow fever is not prevalent in the developed world, we demonstrate that within the yellow fever vaccine response of a HLA B*57:01 positive individual we can detect a breadth of CD8+ T-cell clonotypes that recognise both the yellow fever wildtype KF9 epitope and synthetic variants of this epitope that can only be presented in an abacavir treatment dependent manner. We offer this is as “proof in principle” of an altered peptide repertoire / heterologous immunity model in which the abacavir associated shift in self-peptide presentation contains self-peptide(s) that can cross-react with a pre-existing memory T-cell population.Immunologically confirmed abacavir HSR only occurs in individuals with the HLA-B*57:01 allele and this 100% negative predictive value has been crucial to the success and implementation of HLA-B*57:01 as a routine screening tool to prevent abacavir HSR. However, only 55% of individuals with HLA-B*57:01 exposed to the drug will develop hypersensitivity . We and The authors acknowledge the recently published Minimum Information about T cell Assays (MIATA) Guidelines ,17 and hUnpublished data from the PREDICT-1 study  was anal7/ml in 90% FCS/10% DMSO, transferred into-1C/min freezing containers  at-80 C and then transferred to vapour-phase liquid nitrogen storage, at -180°C until use. Cell numbers and viability were evaluated using an automated cell counter  and average cell yield following thawing was typically 9 × 106 PBMC with 95% viability. The HLA-B*57:01-transfected, MHC-I-deficient B lymphoblastoid cell line C1R.B57 [Healthy or HIV+ donors were selected on the basis of their carriage or not of the HLA-B*57:01 allele. PBMC were separated by density centrifugation  and cryopreserved by suspension at 1 × 10 C1R.B57  was cult5 viable cells in triplicate. Assay plates were evaluated using an automated plate reader .IFN-γ ELISpot assay: Interferon-γ ELISpot assays were performed as previously described , with th2 for six hours, in the presence of 20 μg/mL Brefeldin-A (Sigma-Aldrich) for the final four hours. Samples were surface labelled for CD4 and CD8 expression on ice and then fixed and permeabilized for the detection of intra-cellular IFN-γ, as previously described [Abacavir responsive short term lines were produced from cryopreserved PBMC, as previously described . To enumInterferon-γ (IFN-γ) positive abacavir reactive cells were detected in PBMC from abacavir unexposed donors following stimulation with C1R.B57 APC treated ± abacavir, as described above, for four hours. Cells were then labelled with an IFN-γ capture reagent and IFN-γ was captured for forty-five minutes, according to manufacturer’s directions (Miltenyi Biotec). Cells were then washed and labelled with anti-IFN-γ-PE, anti-CD3-Pacific Blue and anti-CD8-APC-H7 (Becton Dickinson). Cells were either analyzed immediately by flow cytometry or following enrichment of positive cells using anti-PE microbeads and magnetic column separation (Miltenyi Biotec) and then analyzed by flow cytometry . A lymphCryopreserved PBMC samples were labelled using CD4-PE, CD8-APC-Cy7, CD45RA-APC  and CD62L-ECD (Beckman Coulter) on ice, following overnight rest to restore CD62L expression  and two synthetic variants, K9A (KTWGKNLVA) and K9V (KTWGKNLVV), were synthesized by standard 9-fluorenylmethyloxycarbonyl (FMOC) chemistry and purified by reversed-phase high-performance liquid chromatography  . Tetramers were produced as previously described [A-ABC-B*57:01 and K9V-ABC-B*57:01. Successfully folding of MHC-I monomers with these peptides was strictly abacavir dependent. To tetramerize the complexes Streptavidin-R-Phycoerythin (PE), Streptavidin-Allophycocyanin (APC), or Streptavidin-Brilliant Violet 421 (BV421)  was sequentially added over 60 min at a molar ratio of Streptavidin to peptide-MHC-I monomer of 1:4.The yellow fever specific peptide epitope K9escribed  with theF epitope. Cells were harvested day 7 and stained with K9F-B*57:01-PE, K9A-ABC-B*57:01-APC, and K9V-ABC-B*57:01-BV421 tetramers at RT for 20min, and subsequently washed and stained for CD3 and CD8 for 30 min. at 4°C. The cells were subsequently analyzed by flow cytometry .PBMC’s from an HLA-B*57:01 positive donor drawn day 17 post yellow fever vaccinated  were expanded with the K9Statistics: Positivity of abacavir ELISpot responses was defined using a cut-off response of ≥ 10 SFU/106 PBMC above background, and differences in proportions of positive responses were assessed using a Fisher’s exact test. A donor-stratified Wilcoxon test was used to assess paired differences in magnitude of responses across restimulated abacavir specific cell line cultures, p values of less <0.05 were considered significant. Statistical testing was not carried out if the sample size was less than five.The Institute for Immunology and Infectious Diseases (IIID) laboratories at Murdoch University are accredited by the Australian National Association of Testing Authorities (NATA). All assays are performed according to standard operating procedures that are managed using a document control system (Q-Pulse). Laboratory staff training is regularly updated and documented by Q-Pulse. Experimental samples and data are tracked through the laboratory using a Laboratory Information Management Systems (LIMS) and data and sample integrity is protected.Murdoch University, Royal Perth Hospital and Australian Bone Marrow Donor Registry ethics committees approved the study that was conducted according to the principles expressed in the Declaration of Helsinki. All study participants provided informed written consent prior to their participation. A patient consent form approved by the Murdoch University, Royal Perth Hospital and Australian Bone Marrow Donor Registry ethics committees was used to document consent.Abacavir patch testing is a specific and highly sensitive research tool used to identify HLA-B*57:01 positive patients who have experienced immunologically-mediated abacavir HSR ,11,22. TNon-covalent binding of abacavir to HLA-B*57:01 results in the presentation of self-peptides that would not otherwise be presented ,13 suggeWe examined whether the rapid and early clinical responses to abacavir in some patients could be explained by expansion of pre-existing memory CD8+ T cells by exposing PBMC from abacavir-unexposed HLA-B*57:01 positive (n = 3) and negative donors (n = 2) to APC stimulation with either the HLA-B*57:01 transfected single HLA antigen cell line C1R.B57 or autologous PBMC, ± abacavir . We usedin vitro 10 day cultures of PBMC from drug naïve HLA B*57:01 positive donors (2). We have used this culture protocol to examine the contributions of pre-existing memory T cells, and/or naïve T cells in the establishment of abacavir associated immunity, using abacavir-unexposed HLA-B*57:01 positive and negative PBMC from healthy donors, which included three of the four donors examined in We have previously demonstrated that robust abacavir treatment specific CD8+ T-cell responses can be reproducibly induced in Short-term abacavir responsive T-cell lines were generated from all eight unsorted PBMC samples tested. Similar to previous reports ,23, we cin vitro expansion of abacavir specific CD8+ T-cell responses in 100% (8/8) of HLA-B*57:01 positive healthy donors is higher than the 55% incidence of HSR seen in HLA-B*57:01 positive patients exposed to abacavir in the PREDICT-1 trial [These results also confirmed the HLA-B*57:01 restriction of the CD8+ T-cell responses induced in the presence of abacavir . The in -1 trial   with or without abacavir pre-treatment, cultured them for 10–12 days in the presence of IL-2 and then re-stimulated the cultures with the same APC combinations. In cultures derived from sorted memory T cells there were significantly higher %CD8+/ IFN-γ T cells detected when re-stimulated with C1R.B57.ABC APC compared to untreated C1R.B57 APC , which were then stimulated, cultured for 10 days, and then re-stimulated, as described in F (KTWGKNLVF). With frequencies ranging from 0.2–0.6% of the CD8+ T cells, and 1–8% of the activated CD38+, CD8+ T cells, all yellow fever vaccinated HLA-B*57:01 positive donors (n = 6) recognized this wild type K9F epitope [F variants, which could only be presented in the presence of abacavir. To this end, synthetic variants of the K9F epitope were generated containing valine or alanine instead of a phenylalanine at P9 of the epitope. These variant peptides only bound to HLA-B*57:01 in the presence of abacavir [To support the idea that the CD8+ T cells, which can be expanded in the presence of abacavir, could, at least in part, be derived from pre-existing memory T cells, we developed a model system involving human CD8+ T cell responses against a viral target, a live-attenuated yellow fever virus vaccine. A panel of yellow fever virus vaccinees, were used to identify a dominant HLA-B*57:01-restricted, yellow fever-specific CD8+ T cell epitope, K9 epitope . We thenabacavir . Thus, iF-B*57:01 CD8+ T-cell response was polyclonal and could be separated into at least six populations based on T-cell cross-recognition of the abacavir bound variants, K9A-ABC-B*57:01 and K9V-ABC-B*57:01. The first population  or valine (K9V). We generated MHC-I tetramers containing the wild type K9F peptide and MHC-I tetramers containing the K9A or K9V peptides that could be folded only in the presence of abacavir. In both HLA-B*57:01 positive, yellow fever vaccinated donors who were examined, we were able to show that the donor’s memory responses included a breadth of CD8+ T cell clonotypes that included T-cell receptors (TCR) which exclusively recognized the K9F epitopes but importantly also TCR which recognized both the K9F epitope and which also cross-recognized the synthetic variants of the epitope with an alanine (K9A) or valine (K9V) at the P9 position of the epitope.Abacavir binding to HLA-B*57:01 favors the presentation of peptides with an aliphatic C-terminal residue such as valine, alanine or isoleucine rather than the aromatic residues tryptophan or phenylalanine preferred by HLA-B*57:01. We describe a novel HLA- B*57:01 restricted epitope derived from Yellow Fever, K9F and exploited the knowledge of the effect of abacavir on peptide selection to produce C-terminal variants of the epitope containing either an alanine . This mechanism of abacavir-dependent cross-reactions is reminiscent of the “molecular mimicry” that has been invoked in the generation of autoimmune responses. We have previously noted that the alteration of the HLA-B*57:01 bound peptide repertoire caused by abacavir and the resulting massive activation of T cells has parallels with an alloreactive response (12). In combination, these two mechanisms may explain how abacavir can induce a large and diverse population of autoreactive T cells.Similarly, we propose that pre-existing abacavir reactive CD8+ memory T cells are pathogen specific and HLA-B*57:01 restricted. Some of these memory T cells have the potential to cross-react with the many host cells which express abacavir dependent self-peptide/s bound to HLA B*57:01, but only when pharmacologic levels of abacavir are present. It follows that whilst the patient is receiving abacavir treatment, the abacavir dependent self-peptide epitope has the potential to be expressed on a much larger proportion of host cells than those infected by the priming pathogen, an effect which is rapidly lost following drug withdrawal as it is rapidly cleared.ex-vivo from the PBMC of 100% of HLA-B*57:01 positive abacavir unexposed individuals. Since it is likely that HLA-B*57:01 positive abacavir tolerant patients will present the same wide range of novel endogenous epitopes as HSR patients, this suggests that only a subset of the abacavir reactive CD8+ T cells that are detected or expanded in vitro are mediating HSR in vivo [in vitro to a range of neo-antigens to which the T cells have not been tolerized. Only a subset of these allo-responses are believed to mediate organ rejection, and these responses are mediated by pre-existing effector memory CD8+ T cells specific to prevalent and/or persistent viruses [Despite the 55% PPV of HLA-B*57:01 for abacavir HSR, we were able to expand abacavir reactive CD8+ T-cell responses  in vivo ,5,12,13. viruses . Memory  viruses –27. In t viruses –30. In k viruses .in vitro culture system. Alternatively, it has recently been reported that stem cell memory CD8+ T cell (TSCM), restricted to common recall antigens, express CD45A and CD62L+ [SCM may have been included along with truly naïve T cells in our sorts. Like conventional memory T cells, TSCM respond to re-stimulation with cognate antigen rapidly, but have greater proliferative responses [SCM which cross-react to an abacavir dependent self-antigen might explain the incomplete positive predictive value of clinical abacavir HSR, however we did not look for abacavir responsive CD62L+, CD45RA+, CD95+, CD8+ T cells in this study.Whilst we were able to expand abacavir reactive CD8+ T cells from the sorted memory T cells of all our healthy HLA-B*57:01 positive abacavir unexposed individuals, this was true for only about 50% of sorted naïve T cells from the same individuals. The inefficiency of this process may be related to the limitations of our d CD62L+  and we cesponses . Inter-pOther explanations can be offered to explain the incomplete positive predictive value of HLA-B*57:01 for abacavir HSR. Individuals may lack naïve T-cell specificities capable of responding to the new drug peptide target as has been recently demonstrated for HLA-B*15:02 associated Stevens-Johnson syndrome/toxic epidermal necrolysis where a specific TCR clonotype is prevalent amongst HLA-B*15:02 positive cases but not in HLA-B*15:02 positive carbamazepine tolerant controls . AlternaThe non-covalent binding of drug to the MHC molecule resulting in presentation of an altered peptide repertoire provides a mechanistic basis for drug-MHC-peptide interactions and explains how drug hypersensitivity could be exclusively T-cell mediated. The additional contribution of heterologous immunity to this or other models of drug hypersensitivity would explain both the distribution and type of clinical manifestations and the sometimes very rapid onset of symptoms after drug initiation as well as whether or not a drug hypersensitivity syndrome will occur in a genetically predisposed individual. For example, following abacavir exposure in an HLA-B*57:01 positive individual, if cross-reactive CD8+ memory T cells exist in a high enough precursor frequency they will expand inducing rapid onset of symptoms within five days of first drug exposure. In other individuals it is possible that the contribution of these pre-existing cross-reactive memory CD8+ T-cell responses may be slow to expand or weaker than the priming of naïve CD8+ T-cell responses against a broader range of abacavir dependent endogenous peptides, leading to the onset of HSR symptoms in the second to third week of abacavir treatment.In vivo, as in the transplantation model, HLA-B*57:01 restricted CD8+ T-cell responses to a highly prevalent and ubiquitous chronic persistent infection, such as one of the HHV, may prove to be the source of the cross-reactivity to the abacavir-induced neo-antigen. Characterizing the role of such heterologous immune responses in drug hypersensitivity may provide approaches to examine the role of heterologous immunity in other forms of autoimmune or allergic hypersensitivity.If this model is correct then it will be necessary to identify both the relevant specific HLA-restricted anti-pathogen response and the drug-specific self-peptide that the CD8+ T cells are cross-reacting to. T-cell responses to viruses, particularly the chronic persistent human herpes virus (HHV) infections, have been shown to mediate allogeneic organ rejection via TCR cross-reactivity , and we propose a similar heterologous immunity model to explain T-cell mediated drug hypersensitivity. Our observation that T cells recognizing a yellow fever vaccine epitope cross-react with synthetic variants of the epitope that can only be presented by HLA-B*57:01 in the presence of abacavir provide a proof of principle for such a model. However, we emphasize that our experiments were designed only as a proof of principle to show that cross-reactivity can occur and yellow fever vaccination is not a pre-condition for abacavir hypersensitivity. S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FileFigure A. CD62L expression on thawed cryopreserved peripheral blood mononuclear cells. Figure B. Sorted memory phenotype T cells respond to CEF peptide stimulation but naïve cells do not.(DOCX)Click here for additional data file.


Background: PRMT1 is up-regulated in lung cancer.Results: PRMT1 is a novel regulator of EMT and Twist1 is a new PRMT1 substrate.Conclusion: PRMT1-methylation of Twist1 is required for active E-cadherin repression.Significance: Targeting PRMT1-mediated Twist1 methylation might represent a novel strategy for developing new anti-invasive/anti-metastatic drugs. Protein arginine methyl transferase 1 (PRMT1) was shown to be up-regulated in cancers and important for cancer cell proliferation. However, the role of PRMT1 in lung cancer progression and metastasis remains incompletely understood. In the present study, we show that PRMT1 is an important regulator of epithelial-mesenchymal transition (EMT), cancer cell migration, and invasion, which are essential processes during cancer progression, and metastasis. Additionally, we have identified Twist1, a basic helix-loop-helix transcription factor and a well-known E-cadherin repressor, as a novel PRMT1 substrate. Taken together, we show that PRMT1 is a novel regulator of EMT and arginine 34 (Arg-34) methylation of Twist1 as a unique “methyl arginine mark” for active E-cadherin repression. Therefore, targeting PRMT1-mediated Twist1 methylation might represent a novel strategy for developing new anti-invasive/anti-metastatic drugs. Moreover, methylated Twist1 (Arg-34), as such, could also emerge as a potential important biomarker for lung cancer. Lung cancer is the deadliest of all the cancers. High mortalities in lung cancer patients can be partially attributed to (a) difficulties in detecting the disease early, and (b) resistance to currently available standard therapies. Hence, there is a large need for the development of novel strategies to combat this deadly disease. Many cancers display genetic and epigenetic alterations, which strongly contribute to cellular transformation via chromatin remodeling, histone modification, and gene expression; all of these contribute to the gene silencing of tumor suppressors, and the gain of oncogenes. While DNA methylation and chromatin remodeling are considered to be major epigenetic regulators, emerging data implicate protein arginine methylation  as a novel epigenetic regulator in cancer , 2.i.e. PRMT1, PRMT3, PRMT4/CARM1, PRMT6, PRMT8), type II enzymes , and type IV (i.e. PRMT2) enzymes , RNA-binding proteins , transcription factors (e.g. FOXO), and protein translation machinery  are often targets for PRMT-mediated methylation. While histones constitute a class of well-defined PRMT substrates, there is an increasing list of non-histone PRMT substrates, which includes tumor suppressors . PABP1, –15).15.e.g. pPRMT-mediated methylation regulates many essential cell functions mainly through the modulation of protein function, gene expression, and/or cellular signaling. In general, arginine methylation of proteins will have a positive or negative impact on the interactions with other molecules, which could be either other proteins or nucleic acids (DNA or RNA). These altered interactions act as “molecular switches,” which affect either the sub-cellular localization of proteins and/or the stability of protein, or RNA; ultimately affecting either gene expression or cellular signaling.Deregulation of PRMT expression, predominantly up-regulation, has been attributed to several cancers , 16, 17.In the current study, we identified PRMT1 as a novel regulator of Epithelial-Mesenchymal-Transition (EMT), an essential process during cancer progression, and metastasis. Interestingly, the overexpression of PRMT1 in a non-transformed bronchial epithelial cell line resulted in the induction of EMT, characterized by a decrease in E-cadherin, and an increase in N-cadherin expression. Using a complementary approach, we also show that the gene silencing of PRMT1 in non-small cell lung cancer (NSCLC) cell lines lead to the reversal of EMT. Furthermore, PRMT1-mediated effects were not solely restricted to E- to N-cadherin switching. PRMT1 gene silencing in NSCLC cells also induced the formation of spheroids when cultured in Matrigel, and reduced migration and invasion, characteristics of epithelial cell phenotype. Moreover, we also identified Twist1, an important E-cadherin repressor, as a novel PRMT1 substrate and PRMT1-mediated methylation of Twist1 at arginine 34 (Arg-34) as an important event for E-cadherin repression. Thus, PRMT1 is shown to be a novel regulator of EMT and PRMT1 methylation of Twist1 at arginine 34 (Arg-34) as a unique “methyl arginine mark” for active E-cadherin repression.Mouse Twist1 was obtained from Addgene (plasmid number 1783) and was engineered in-house into pCMV-Myc and pGEX-4T1 vectors. Site-directed mutagenesis was performed using QuickChange Site-directed Mutagenesis kit (Stratagene). Human PRMT1 cDNA sequence was subcloned into pCMV-HA vectors in-frame with HA tag sequence.2 incubator at 37 °C. Whereas MCF7 cells were cultured in DMEM medium supplemented with 10% FBS, in a humidified 5% CO2 incubator at 37 °C. All the cell lines were cultured bi-weekly and stocks of cell lines were passaged no more than ten times for use in experiments.Human non-transformed bronchial epithelial cell line (Beas2B), NSCLC cell lines , and human breast cancer cell line (MCF7) were obtained from the tissue culture core of the University of Colorado, Anschutz Medical Campus. Beas2B, A549, and H2122 were cultured in RPMI medium supplemented with 10% FBS, in a humidified 5% COFor generating A549 and H2122 clones with stable expression of non-targeting shRNAs and PRMT1 shRNAs, A549 and H2122 cells were transfected with either pENTR/H1/TO-control shRNA or pENTR/H1/TO-PRMT1 shRNA vectors followed by 200 μg/ml and 100 μg/ml Zeocin selection, respectively. Several zeocin-resistant clones were subsequently isolated and screened for PRMT1 knockdown via immunoblotting.et al., (H2122 clones were grown in growth factor reduced Matrigel (BD Bioscience) basement membrane according to Debnath et al., . BrieflyCells were grown to confluence in complete cell culture medium. At time 0, a 3-mm scrape wound was created across the diameter with a pipette tip followed by extensive washes with medium to remove dead and floating cells. After adding complete medium supplemented with 1 μg/ml Mitomycin C, cell migration was recorded at 0, 12, 24, and 48 h. Images were captured using an inverted microscope equipped with a digital camera. Images were later analyzed by determining the distance between the cells on either side of the scratch overtime, and are represented in the figure as percent scratch closure.For assessing cell migration, 30,000 cells in serum free media were seeded into the transwell inserts (Corning) containing 8-μm permeable pores and allowed to migrate toward 10% FBS-containing medium. Later, the cells in the transwell inserts were removed, and the inserts were washed in PBS three times. The migrated cells on the bottom of the insert were fixed with 2% glutaraldehyde solution followed by crystal violet (1%) staining. After washing the inserts three times with PBS, the inserts were allowed to air dry and pictures were taken using an inverted microscope. Ten independent fields were counted for each transwell and the average number of cells/field were represented in the graphs. For assessing cell invasion, 30,000 cells in serum-free medium were seeded in the matrigel-coated transwell inserts (BD Bioscience). The cells were later processed similar to that of cell migration assay.Soft agar assays were performed as described previously . Briefly6 in 100 μl of PBS) were injected into the later tail veins of athymic nude mice (Harlan Laboratories). Mice were sacrificed after 8 weeks, and lung tumors were counted. Later, lungs were inflated in situ with PBS: Tissue-Tek O.C.T. compound (50:50) by intra-tracheal intubation, flash frozen, and embedded in Tissue-Tek O.C.T. compound. Hematoxylin and Eosin (H&E) staining was performed on the lung sections, and stained sections were later scanned using Aperio Scanscope CS and its associated Spectrum® image Management and Analysis system.Animal experiments were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol (13–016) was approved by the Institutional Animal Care and Use Committee (IACUC). H2122 clones with stable expression of non-targeting shRNAs and PRMT1 shRNAs were detached from the culture dish, collected by centrifugation, and re-suspended in PBS. For the lung metastasis assay, cells , at 30 °C for 1 h. After 1 h, the reactions were stopped and separated on a SDS-PAGE gel. The separated proteins were later transferred to nitrocellulose membrane, amplified , dried, and fluorography was performed.eviously , 13, 20.IIF studies were performed as described earlier . Brieflym siRNAs by using Lipofectamine 2000 reagent according to the manufacturer's protocol. For generating stable clones, shRNA constructs were made by cloning the above PRMT1 siRNA sequence oligos into pENTR/H1/TO plasmid (Life Technologies).Double-stranded RNAs (siRNAs) targeting human PRMT1 (5′-GACUUCACCAUCGACCUGGACUUCA-3′), and control siRNAs (5′-UCUGUGAUUUGAAAGACUAGCCAAG-3′) were procured from Invitrogen  and Santa Cruz Biotechnology (SC-41069), while siRNAs targeting human Twist (SC38604) and control siRNA (SC-37007) were obtained from Santa Cruz Biotechnology. NSCLC cells were treated with 50 nm β-glycerophosphate, pH 7.20, 0.1 mm sodium vanadate, 2 mm MgCl2, 1 mm EGTA, 1 mm dithiothreitol, 2 μg/ml leupeptin, and 4 μg/ml aprotinin). The following antibodies were used for immunoblotting: anti-E-cadherin and anti-N-cadherin (BD Bioscience), anti-PRMT1 , anti-Myc and anti-actin (Sigma Aldrich), and anti-HA (Roche Life Sciences). Aliquots of various extracts were resolved on 10–12% SDS-PAGE gels and transferred to nitrocellulose membranes. The filters were blocked in Tris-buffered saline  with 3% nonfat dry milk and then incubated with the same blocking solution containing the indicated antibodies at 0.5 μg/ml for 12–16 h. The filters were extensively washed in TTBS, and bound antibodies were visualized with horseradish peroxidase (HRP)-coupled secondary antibodies.Cell extracts were prepared in a lysis buffer  as per the manufacturer's recommendations. For quantitative RT-PCR, 5 μg of total RNA was reverse transcribed using random primers and real-time PCRs were performed using the QuntiTect SYBR Green PCR kit  and the Bio-Rad CFX real-time PCR detection system. The primers utilized in the PCR experiments were as follows; hPRMT1- F 5′-CCTTCACCTCCCCGTTCTG-3′, R 5′-CCAGGGCGTGCACGTAGT-3′, hTwist1-F 5′-GCGCTGCGGAAGATCATC-3′, R 5′-GGTCTGAATCTTGCTCAGCTTGT-3′.t test for assessing variance. Increase in statistical significance  is denoted with an “*” symbol, while a decrease in statistical significance  is denoted with a “#” symbol.Data were compiled from at least three independent, replicate experiments, each performed on separate cultures and on separate occasions. The responses are displayed as “fold-changes.” Comparisons of data among experimental groups were performed using Student's i.e. Beas2B). The overexpression of PRMT1 was ascertained by immunoblotting with anti-PRMT1 and anti-HA antibodies (A). Interestingly, overexpression of PRMT1 induced a dramatic decrease in E-cadherin (A). Additionally, we also observed a remarkable increase in N-cadherin, a well-known mesenchymal cell marker (A). We next tested the effects of PRMT1 knockdown on EMT. For these studies, we designed and tested two different small interference RNA sequences (siRNAs) specific to PRMT1. Treatment of NSCLC cell lines (i.e. A549 and H2122) with PRMT1-specific siRNAs suppressed the expression of PRMT1 by more than 70% (B–E). Notably, PRMT1 knockdown reversed EMT, as defined by an increase in E-cadherin expression and a decrease in N-cadherin expression (B–E).PRMT1 expression was shown to be up-regulated in cancers and important for cancer cell proliferation , 21. Howtibodies A. Interecadherin A. Additil marker A. We nexthan 70% , B–E. Nopression , B–E.A and B). Consistent with the effects of PRMT1 specific siRNAs on EMT, multiple A549 and H2122 clones with stable knockdown of PRMT1 also showed an increase in E-cadherin expression as determined by Western blots (A and B) and indirect immunofluorescence (C and D). Furthermore, we also observed a striking decrease in N-cadherin expression (A and B). The expression of EMT markers in stable cells (A and B) is different from that of parental cell lines (B–D), since the stable cell lines were homogenous as they were derived by single cell isolation technique. Interestingly, stable knockdown of endogenous PRMT1, in addition to augmenting E-cadherin expression, also induced the formation of spheroids when embedded in Matrigel matrices as single cells (E). Similar results were obtained with A549 clones stably expressing PRMT1 shRNAs (data not shown). Taken together, these data suggest that PRMT1 plays an important role in the regulation of E-cadherin.Three-dimensional cell cultures are useful tools to examine the epithelial phenotype of cells wherein the epithelial cells would form spheroids , 23. To rn blots , A and Brescence , C and Dpression , A and Ble cells , A and Bll lines , B–D, sile cells E. SimilaA). For these assays, a 3-mm scrape wound was created on confluent cultures of H2122 clones expressing either non-targeting shRNAs or PRMT1 shRNAs. The ability of the cells to migrate and heal the wound was recorded over a period of time in the presence of the mitosis blocker (Mitomycin C). H2122 clone expressing PRMT1 shRNAs displayed reduced motility in comparison to H2122 clone expressing non-targeting shRNAs . We have also tested the migratory potential of H2122 and A549 clones by using a trans-well assay (B and C). For these assays, cells in serum free medium were seeded into the trans-well inserts and allowed to migrate through a permeable membrane (8 μm) toward 10% serum containing medium. The migrated cells on the bottom of the insert were later fixed, stained, counted and are represented in the graphs (B and C). Interestingly, suppression of PRMT1 expression was associated with reduced cell migration in both A549 and H2122 clones (B and C). Conversely, over expression of PRMT1 in a non-transformed bronchial epithelial cell line (Beas2B) was found to increase cell migration .We explored next if PRMT1 could also affect cell migration and invasion . For tesFig. 3E and F). A549 and H2122 clones expressing PRMT1 shRNAs also showed reduced invasion when compared with clones expressing non-targeting shRNAs (E and F). These data suggest that PRMT1 is a novel regulator of cell migration and invasion.To measure the invasive abilities of A549 and H2122 clones expressing PRMT1 shRNAs, we performed invasion assays. In these assays, cells in serum free media were seeded into the trans-well inserts coated with a reconstituted basement membrane gel . The cells that have invaded the Matrigel and migrated through the pores were later fixed, stained, counted and are represented in the graphs , E and Fg shRNAs , E and Fin vivo, H2122 cells stably expressing either PRMT1 shRNAs or non-targeting control shRNAs were injected into the lateral tail-veins of athymic nude mice, and the formation of lung tumors were examined. Consistent with the effects of PRMT1 knockdown on EMT, migration, and invasion, we observed a striking reduction in the number of lung tumors formed in mice injected with H2122 cells stably expressing PRMT1 shRNAs (G). We also confirmed that the effects of PRMT1 knockdown on the metatstatic potential of H2122 cells were not due to the induction of apoptotic cell death via AnnexinV/propidium iodide staining (data not shown). Thus, PRMT1 appears to act as an important regulator of tumor metastasis in lung cancer, and the effects of PRMT1 knockdown on tumor metastasis were not due to apoptotic cell death.To test the effects of PRMT1 knockdown on the metastatic potential of H2122 cells 1 shRNAs G. We alsA and B). Remarkably, the knockdown of PRMT1 significantly attenuated colony formation of both A549 and H2122 clones expressing PRMT1 shRNAs compared with the clones with stable expression of non-targeting control shRNAs (A and B). These results suggest that PRMT1, in addition to regulating the induction of EMT, cell migration, and invasion, also plays an important role in the transformation of cells.The soft agar colony formation assay is a common method to measure anchorage-independent growth. To determine if PRMT1 also plays a role in transformed cell growth, A549 and H2122 clones stably expressing either PRMT1 shRNAs or non-targeting control shRNAs were seeded into soft agar cultures , A and Bl shRNAs , A and Bin silico analysis of primary amino acid sequences of known E-cadherin repressors  revealed Twist1 as a potential PRMT1 substrate (A). Twist1 displayed two potential PRMT methylation (RG) sites  that were well conserved across the species, suggesting that the arginine methylation sites might play an important role for Twist1 function (B).Since PRMT1 is a novel regulator of E-cadherin  and 2, wubstrate A. Twist1function B.in vitro methylation assay (C and D). For these assays, HA-tagged PRMT1 was expressed and purified from non-transformed bronchial epithelial cells  or NSCLC cells . Recombinant Twist1 (GST-Twist1) was expressed and purified from Escherichia coli. The in vitro methylation reaction included the isolated PRMT1, GST-Twist1 and S-adenosyl l-methionine (SAMe) as a methyl donor. PRMT1 catalyzed strong methylation of Twist1 (C and D).To test directly if Twist1 was indeed a PRMT substrate, we performed an on assay , C and DA and B). We next tested the specificity of PRMT1 to the predicted arginine residues (Arg-34 or Arg-74). For these experiments, we made methylation-deficient mutants of Twist1 by substituting arginines to similarly charged lysines . Interestingly, PRMT1 efficiently catalyzed the methylation of wild-type and R74K mutant of Twist1 (E), but not the R34K mutant (E). Taken together, these results indicate that Twist1 is a novel PRMT1 substrate and PRMT1 predominantly catalyzes Arg-34 methylation of Twist1.Twist1 encodes two potential PRMT methylation sites , we made use of methyl-arginine specific antibodies . Pull downs performed on cells transfected with Myc-Twsit1 displayed methylation of Twist1 . Pull downs performed on cells transfected with empty vector, on the contrary, failed to show methylation and served as negative control . Additionally, in vivo methylation of Twist1 was severely attenuated in the absence of PRMT1 expression, indicating the specificity of the anti-mono methyl-arginine specific antibodies .To determine if Twist1 was methylated endogenously (A). Expression of R34K mutant, in contrast, failed to impact E-cadherin expression , suggesting that the Arg-34 methylation of Twist1 is required for E-cadherin repression. In addition, we also observed a corresponding increase in N-cadherin, a mesenchymal cell marker, by the wild-type Twist1 but not by the R34K mutant of Twist1 . Similar effects of R34K mutant of Twist1 on E-cadherin repression were also observed in H2122 cells (data not shown). We also confirmed the effects of Twist1 expression on E-cadherin repression via indirect immunofluorescence . A549 cells expressing wild-type Twist1  displayed reduced E-cadherin staining, while A549 cells expressing R34K mutant displayed positive staining for E-cadherin . To ensure that the Twist1 arginine methylation-mediated E-cadherin repression was not restricted to the epithelium of the lung, we also tested the effects of wild-type and methylation-deficient mutants of Twist1 on E-cadherin repression in another epithelial cell-derived cancer cell line, MCF7 (a human breast cancer cell line). Consistent with the effects of the Twist1 mutants on E-cadherin repression in NSCLC cells, expression of the R34K mutant also failed to repress E-cadherin in MCF7 cells, as determined by Western blot , and indirect immunofluorescence .Since Twist1 is methylated by PRMT1 specifically at arginine 34 Arg-34, , we sougFig. 6E). Expression of wild-type Twist1 in A549 cells, but not the R34K mutant of Twist1, resulted in increased cell migration (E). These results indicate that the arginine methylation of Twist1 at Arg-34 might be important for Twist1-induced cell migration.We next investigated the effects of Twist1 (WT and R34K mutant) expression on A549 cell migration using trans-well assays E. Expresigration E. These F). Knockdown of Twist1 provoked increased E-cadherin expression (F). While the expression of wild type Twist1 could rescue Twist1 function (as determined by reduced E-cadherin levels), expression of the R34K mutant, on the contrary, failed to repress E-cadherin expression (F). Furthermore, failure of Twist1 to repress E-cadherin in the absence of PRMT1 (G) demonstrates the importance of PRMT1 methylation of Twist1 for E-cadherin repression. Taken together, our results identified a previously unknown functional aspect of the conserved arginine residue within Twist1 in E-cadherin repression. These studies further demonstrate that Twist1 methylation at Arg-34 represents an important “methyl arginine mark” that defines active E-cadherin repressionTo confirm the requirement of Arg-34 methylation for Twist1 function, we also performed “rescue” experiments in the Twist1 depleted (knockdown) A549 cells, employing wild type and R34K mutant of mouse Twist1 F. Knockdpression F. While pression F. Furtheof PRMT1 G demonstH). The Twsit1 levels were normalized against actin . Wild-type Twist1 decayed with a half-life of ∼6.5 h . Interestingly, there is no significant difference in the decay rates of R34K mutant . These results suggest that Arg-34 methylation might not play a role in stabilizing Twist1 protein.Several studies highlight the importance of post-translational modifications, predominantly phosphorylation, in regulating the protein stability of Twist1 –27. To t–Fig. 6B) and MCF7  cells. Expression of R34K mutant of Twist1, on the contrary, was observed to be predominantly cytoplasmic in both A549 and MCF7 cells (B and D). These data would suggest that Arg-34 methylation of Twist1 might have a potential role in the regulation of nuclear import of Twist1.It was shown earlier that arginine methylation of proteins can influence their sub-cellular localization . InteresFig. 6E). As evinced from the works of Bissell, Brugge, and others, transformed cells tend to grow in a disorganized (form aggregates) manner in basement membrane cultures, while non-transformed epithelial cells, on the contrary, establish apical-basal polarity and grow as spheroids , only Twist1 was identified as a PRMT1 substrate. Considering the importance of Twist1 during tumor progression, we strongly believe that our findings will have far-reaching implications in gaining a better understanding of tumor progression in general.e.g. scleraxis, paraxis, and Hand2. Interestingly, paraxis is also shown to specifically bind E-box elements, like Twist1 (Twist1 belongs to a family of basic helix-loop-helix proteins (bHLH), which function as transcription factors . Most im34–38–e Twist1 . HoweverH), indirect immunofluorescence detection of Twist1 expression revealed a possible role for Arg-34 methylation of Twist1 in nuclear import (B and D). Since, basic helix-loop-helix proteins are known to form homo- and heterodimers, we speculate that arginine methylation of Twist1 might trigger homo- and/or hetero-dimerization of Twist1 , as such, could also emerge as a potential important biomarker for lung cancer. Thus, PRMT1 not only represents a unique class of EMT regulators but also represents a potential new class of drug targets for future therapeutic treatments of lung cancer.


The VEGF in low oxygen conditions are reported to prolong the survival of malignant cell, and thus this gene has a critical role in tumor growth and invasion as well as development of malignant tumor. We aimed to assess the association between the six common SNPs and the risk of osteosarcoma, and their association with environmental factors.176 subjects with osteosarcoma and 176 gender- and sex-matched healthy control individuals were enrolled into our study. The VEGF -2578C/A, -1156G/A, +1612G/A, +936C/T, -634G/C and -460T/C gene polymorphisms were determined using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay according to manufacturer’s instructionsBy conditional logistic regression analysis, AA and CA+AA genotypes of VEGF -2578C/A were associated with significant increased risk of osteosarcoma compared with CC genotype, and the ORs(95%CI) were 2.32(1.18-4.60) and 1.68(1.07-2.64), respectively. Moreover, individuals with CC and TC+CC genotypes of VEGF-460T/C had significant increased risk of osteosarcoma compared with those carrying with the TT genotype, and ORs(95%CI) were 2.15(1.10-4.21) and 1.60(1.0-2.58), respectively. By stratified analysis, we did not find statistically significant associated between VEGF -2578C/A and -460T/C gene polymorphisms and cancer risk by stratification analysis.Our results suggested that VEGF -2578C/A and -460T/C gene polymorphisms may be association with an increased risk of osteosarcoma. Osteosarcoma is the most common type of primary bone cancer, and this cancer is a malignant tumor arising from mesenchymal tissues. Osteosarcoma mostly occurs in the long bones of the body, such as distal and proximal tibia and proximal humerus.9It is well known that vascular endothelial growth factors (VEGF), a potent angiogenic growth factor, plays an important role in altering proliferation to inflammatory and ischemic processes.The VEGF in low oxygen conditions are reported to prolong the survival of malignant cell, and thus this gene has a critical role in tumor growth and invasion as well as development of malignant tumor.In the present hospital-based case-control study, 176 subjects with osteosarcoma and 176 gender- and sex-matched healthy control individuals were collected from the Department of Orthopedics of Beijing Hospital between May 2011 and December 2013. The osteosarcoma patients were newly diagnosed and histologically confirmed by two pathologists. The control subjects were randomly selected from a pool of individuals who were attending a clinic for routine examination during the same period. The controls were frequency matched to the cancer cases by age and gender, and the control subjects were free from any cancer or other chronic disease, and unrelated to the patients.The written informed consent before participating into this study was obtained from all study participants, and this study was approved by the ethics committee of the Department of Orthopedics of Beijing Hospital.The general demographic and clinical characteristics of osteosarcoma cases and control subjects were collected from medical records, including age, gender, family history of cancer, tumor location, therapy and stage of tumor.Peripheral venous blood was collected from each osteosarcoma case and control subject. According to the manufacturer’s instructions, genomic DNA was extarcted from collected peripheral blood mononuclear cells using TIANamp Blood DNA Kit . The VEGF -2578C/A, -1156G/A, +1612G/A, +936C/T, -634G/C and -460T/C gene polymorphisms were determined using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay according to manufacturer’s instructions . The PCR primers of VEGF -2578C/A, -1156G/A, +1612G/A, +936C/T, -634G/C and -460T/C were designed by Sequenom Assay Design 3.1 software . The PCR reaction conditions were as follows: 95 °C for one minute, then 40 cycles of 95 °C for 20 sec, 60 °C for 1 min and 72 °C for 1 min, and a final extension step at 72 °C for 10 min. The digested PCR products were visualized by ultraviolet (UV) light.2-test. The Hardy–Weinberg equilibriums of VEGF -2578C/A, -1156G/A, +1612G/A, +936C/T, -634G/C and -460T/C genotype frequencies in controls were tested using goodness-of-fit χ2-test. The odds ratio (OR) and their corresponding 95% confidence intervals (CI) for the association between the six VEGF gene polymorphisms and osteosarcoma risk were assessed by conditional logistic regression. Interaction between genotypes of the six VEGF gene polymorphisms and general demographic characteristics was assessed by conditional logistic regression. All P-values were two sided, and a P-value less than 0.05 was considered as statistical significant. All analyses were performed using SPSS version 16.0 statistical software .Comparison of general demographic and clinical characteristics between groups was performed using χSelected demographic and clinical characteristics of the osteosarcoma patients and controls are shown in P>0.05), but the genotype frequencies of +936C/T were not (P<0.05). By conditional logistic regression analysis, AA and CA+AA genotypes of VEGF -2578C/A were associated with significant increased risk of osteosarcoma compared with CC genotype, and the ORs(95%CI) were 2.32(1.18-4.60) and 1.68(1.07-2.64), respectively. Moreover, individuals with CC and TC+CC genotypes of -460T/C had significant increased cancer risk of osteosarcoma compared with those carrying with the TT genotype, and ORs(95%CI) were 2.15(1.10-4.21) and 1.60(1.0-2.58), respectively. However, no significant association was found between -1156G/A, +1612G/A, +936C/T and -634G/C and osteosarcoma risk in multivariate logistic regression analysis.The association of VEGF -2578C/A, -1156G/A, +1612G/A, +936C/T, -634G/C and -460T/C with risk of osteosarcoma are shown in We assessed the association between VEGF -2578C/A and -460T/C gene polymorphisms and risk of osteosarcoma stratified by age, gender and family history of cancer . HoweverIt is reported that many environmental and genetic factors can influence the individuals susceptibility to the carcinogenesis of osteosarcoma.15Several case-controls studies have reported the association of functional polymorphisms in VEGF with increased risk of several tumors, including osteosarcoma.For the association between VEGF gene polymorphisms and development of osteosarcoma, three previous studies have reported their association.Two limitations should be considered in this study. First, the cases and controls were selected from on hospital, and distributions of VEGF +936C/T gene polymorphism in controls were not in line with Hardy–Weinberg equilibrium. The cases and controls may not represent the entire general population. Second, the small sample size of this study may limit the statistical power to find the difference between groups. Therefore, further studies with more different populations and larger sample size could help to verify the role of VEGF gene polymorphisms in the development of osteosarcoma.In this case-control study, our results suggested that VEGF -2578C/A and -460T/C gene polymorphisms may be associated with an increased risk of osteosarcoma. Further large sample studies are greatly needed to confirm these associations.


Recently, T-helper 17 subset (Th17 cells), which produces IL-17A, IL-17F, IL-21, and IL-22, has been implicated in SLE. As CCR6 enriches for Th17 cells, we used this approach to investigate whether CCR6+) (n = 16) and negative (IFN-) (n = 9) patients, as assessed by mRNA expression of IFN-inducible genes (IFIGs) in monocytes. Expression of IL-17A, IL-17F, IL-21, and IL-22 by CD4+CD45RO+CCR6+ T cells (CCR6+ cells) was measured with flow cytometry and compared between IFN+, IFN- patients and HCs.In total, 25 SLE patients and 15 healthy controls (HCs) were included. SLE patients were divided into IFN type I signature-positive –a factor strongly correlating with IFN type I - and IL-21 producing CCR6+ cells.Increased percentages of IL-17A and IL-17A/IL-17F double-producing CCR6+ memory T-helper cells in IFN+ SLE patients, supporting the hypothesis that IFN type I co-acts with Th17 cytokines in SLE pathogenesis.We show for the first time higher percentages of IL-17A and IL-17A/IL-17F double-producing CCR6 Systemic lupus erythematosus (SLE) is a debilitating systemic autoimmune disease characterized by the production of autoreactive antibodies and multiorgan inflammation . A hallm+ T cells (Th17 cells), CD8+ T cells, CD4-CD8-CD3+  T cells, natural killer cells, γδ-T cells, and mast cells [+ T cells differentiate to Th17 cells under the influence of IL-6 and TGF-β [Another key factor in the pathogenesis of SLE, apart from IFN type I, is interleukin-17A (IL-17A) -9. IL-17st cells ,11. Naïvnd TGF-β . The expnd TGF-β ,14. C57Bnd TGF-β . In SLE Co-activity between IFN type I and IL-17/Th17 cells has been suggested in autoimmune diseases ,17. In e-/- mouse model. Ro52/TRIM21 is involved in the ubiquitination of interferon regulatory factors (IRFs), a process that limits the IFN type I response [-/- mice develop an SLE-like phenotype [-/- mouse line, they do not develop SLE, indicating that the development of an SLE phenotype through enhanced IFN type I production in these mice is dependent on the IL-17/Th17 pathway.Co-activity of IFN type I and Th17 pathways has also been suggested for SLE by the Ro52/TRIM21response . After ehenotype . InteresBAFF). BAFF transgenic mice develop lupus-like disease [BAFF protein has been found in SLE patients, correlating with increased disease activity [BAFF mRNA expression in monocytes and the IFN type I signature in primary Sjögren syndrome (pSS) patients [BAFF, has been reported to induce synergistically the differentiation of human memory B cells into antibody-producing plasma cells in the absence of further co-stimulation [BAFF is known to be involved in germinal center formation [Yet another important factor involved in the pathogenesis of SLE is B cell-activating factor of the tumor necrosis factor family  and in sodium-heparin tubes for peripheral blood mononuclear cell (PBMC) preparation, as described previously . CD14-po-ΔΔCT method .Total RNA was isolated from purified monocytes followed by cDNA preparation and real-time quantitative polymerase chain reaction (RQ-PCR) analysis by using predesigned primer/probe sets (Applied Biosystems) . For calPBMCs were restimulated, stained, and measured with flow cytometry, as previously described . For extP < 0.001). Eigenvalues were derived to assess the amount of variance explained by each component factor.The expression levels of 11 IFN type I-inducible genes (previously found to be increased in SLE) were submitted to a principal component analysis to identify correlated groups of genes to reduce data complexity. Kaiser-Meyer-Olkin measure of sampling adequacy was 0.839 with a significant Bartlett test of sphericity , and comparisons were made by using the nonparametric Mann–Whitney IFI44L, IFITM1, SERPING1, and LY6E) to explain 95% of the total variance of the 11 IFN type I-inducible genes within the SLE cohort. Given that the expression of these four IFN type I-inducible genes was not normally distributed, log transformations of expression values were performed, and IFN scores were calculated as described for pSS [HC of each gene in the HC-group were used to standardize expression levels. IFN scores per subject represent the sum of these standardized scores. When we set the threshold for a positive IFN type I signature at IFN score of 8 [In monocytes of 25 SLE patients and 15 HCs, we assessed the expression levels of 11 IFIGs previously assessed in monocytes from patients with primary Sjögren syndrome (pSS)  , and the for pSS . MeanHC ore of 8 , 64% of + cells were selected after gating on lymphocytes and memory Th cells (CD4+CD45RO+ cells) within PBMCs and after CD25+ cells were excluded. To investigate whether the IFN type I signature is associated with an increase in Th17 cytokines expressed by memory CCR6+ T cells, we measured the percentages of IL-17A, IL-17F, IL-22 and IL-21 producing CCR6+ T memory cells in SLE patients positive for the IFN type I signature (IFN+) and patients negative for the signature (IFN-) and HC. The percentages of CCR6+ cells within the CD4+CD45RO+ T-cell population and within total lymphocytes were comparable between the three studied groups (data not shown). Interestingly, the percentages of CCR6+IL-17A+ cells were significantly increased in IFN+ patients, as compared with IFN- patients (P = 0.03), and a higher trend was observed compared with HCs (P = 0.07)   and IFN+ patients versus HC (P = 0.09) . Except for race, no differences in demographic, clinical, or laboratory data were found between IFN+ cells and IFIG expression    with other parameters assessed in this study, we observed a significant positive correlation between the expression of IL-17A and IL-17F within CCR6n Figure A,B. Also) Figure C. No cor) Figure D. Both B+ T memory cells in IFN type I-positive SLE patients. This finding further strengthens the hypothesis that IFN type I and Th17 cells, by co-acting, contribute to the pathogenesis of SLE. Further research to understand the link between these two pathways is warranted.Here we show for the first time a co-occurrence of increased IFN type I activity and increased IL-17/Th17 system in SLE patients. We found increased percentages of IL-17A, IL-17A/IL-17F, and IL-21 producing CCR6+ T cells [A possible mechanism explaining the co-occurrence of IFN type I and IL-17/Th17 immune pathway in SLE could be that both IFN type I and production of IL-6 and IL-23 by DCs are regulated through IRF-5 ,39. Acti T cells .BAFF. BAFF is reported to be involved in DC maturation and DC-driven Th17 cell differentiation in vitro[BAFF gene silencing ameliorated joint pathology and inhibited the generation of Th17 cells in the joints of a collagen-induced arthritis (CIA) mouse model [In addition to the direct effect of IFN type I on Th17 cells, IFN type I may also act indirectly through the production of  in vitro. BAFF gese model . In turnse model , which cBAFF, an IFN type I inducible factor, and the Th17 produced cytokine IL-21. Ettinger et al.[BAFF promotes B-cell responses by bypassing the need for T-cell help or TLR signaling. As these downstream factors are both involved in activation and selection of B cells, these findings again support the concept that IFN type I and the Th17 pathway act together in driving the disease process of SLE.We find a correlation between er et al. showed t+CD45RO+), which include the Tfh cells. The expression of IL-21 by these cells is significantly increased in IFN+ patients compared with IFN- patients and HCs, and IL-21 expression by memory cells also strongly correlated with IFN score (data not shown). These data suggest that Tfh effector function may also be increased in IFN+ patients; however, further studies are required.IL-21 is also produced by T follicular helper (Tfh) cells, and production by Tfh is crucial for B-cell immunity. By gating for CCR6 expression, we exclude the Tfh cells from our analysis. We therefore measured the production of IL-21 by total memory T cells . Unfortunately, we were unable to detect IL-17A and F in most of the samples. We did find IL-22 in serum samples of all subjects, but they were not different between the groups. We also found higher levels of IL-21 in IFNet al.a). Our data indicate that IFN type I might act in concert with Th17 cytokines, paving the way for combination therapies, possibly resulting in more significant clinical effects in the future.Although we do not show a functional link between IFN type I and the Th17 pathway, our findings provide the first support for co-occurrence of increased IFN type I activity and increased IL-17/Th17 system in SLE. The Th17-IFN type I interaction found in this study might have implications for future treatment of SLE and other systemic autoimmune diseases in which IFN type I plays a role. Preliminary results from a phase IIa trial with human IgG1κ anti-IFNα antibody, in 87 SLE patients, showed so far a 40% reduction in IFN type I-induced gene expression but no clinical effect compared with placebo  receptor 6; EAE: experimental autoimmune encephalomyelitis; HC: healthy control; IFIG: IFN-inducible gene; IFN: interferon; IL: interleukin; IRF: IFN-regulatory factor; MS: multiple sclerosis, pSS, primary Sjögren syndrome; NETs: neutrophil extracellular traps; pDC: plasmacytoid dendritic cell; SLE: systemic lupus erythematosus; SLEDAI: Systemic Lupus Erythematosus Disease Activity Index; STAT: signal transducer and activator of transcription; Tfh: T-follicular helper; Th: T helper; TLR: Toll-like receptor.The authors declare that they have no competing interests.ZB and OBJC were involved in the study design and clinical and laboratory data collection, analyzed the data, and drafted and revised the manuscript. CGvH-M, NIM, SMJP, and ND were involved in laboratory data collection and revising the draft article. RJEMD, PLvD,VAD, PMvH, JMWH were involved in collection of clinical data and revising the draft. JPvH was involved in the design of the study and helped to draft the manuscript. MAV and EL were involved in study design, monitoring of data collection, and final draft and approval of the manuscript. All authors read and approved the final manuscript.+ memory T-cell population (defined as CD4+CD45RO+CD25-CCR6+) in PBMCs of IFN negative (IFN-) and IFN positive (IFN+) patients and healthy controls (HCs). (B) Representative graphs of proportions of IL-17A and IL-22-expressing cells within CCR6+ memory T-cell population in PBMCs of IFN- and IFN+ patients and HCs. (C) Representative graphs of proportions of IL-21-expressing cells within CCR6+ memory T-cell population in PBMCs of IFN- and IFN+ patients and HCs.(A) Representative graphs of proportions of IL-17A- and IL-17F-expressing cells within CCR6Click here for file


The viral cell receptors and infection can be blocked by the expression of the viral receptor-binding protein. Thus, the viral cell receptor is an attractive target for anti-viral strategies, and the identification of viral cell receptor is critical for better understanding and controlling viral disease. As a model system for viral entry and anti-retroviral approaches, avian sarcoma/leukosis virus  has been studied intensively and many milestone discoveries have been achieved based on work with ASLV. Here, we used a DF1 cell line expressed viral receptor-binding protein to efficiently identify chicken Annexin A2 (chANXA2) as a novel receptor for retrovirus ALV-J (avian leukosis virus subgroup J). Our data demonstrate that antibodies or siRNA to chANXA2 significantly inhibited ALV-J infection and replication, and over-expression of chANXA2 permitted the entry of ALV-J into its non-permissible cells. Our findings have not only identified chANXA2 as a novel biomarker for anti-ALV-J, but also demonstrated that cell lines with the expression of viral receptor-binding protein could be as efficient tools for isolating functional receptors to identify novel anti-viral targets. The binding of the viral surface protein to the receptors expressed in host cells triggers the viral infection and pathogenesis124569As a model system for viral entry, avian sarcoma/leukosis virus  has been studied intensively, and several important receptors for ASLV entry have been identified by traditional methods111213141617The pcDNA-env_DF1 cell line expressing ALV-J Env protein was previously constructed and shown to be resistant to ALV-J infectionTo further confirm this finding, a recombinant adenovirus rAd-SUJ-rIgGFc expressing fusion protein SUJ-rIgGFc  was consTo test whether chANXA2 serves as a functional receptor for ALV-J infection, we used antibodies against chANXA2 to perform blocking assays to evaluate the effects of chANXA2 on ALV-J infection in DF1 cells. Our results revealed that the viral infection/replication of ALV-J was significantly inhibited in groups that had been treated with anti-ANXA2. As shown in To further evaluate whether the low expression of chANXA2 could confer resistance to ALV-J infection in its susceptible cells, we did siRNA against chANXA2 in DF1 cell, and then tested the effect on the ALV-J infection/replication. As described in To extend our finding and investigate whether the over-expression of the chANXA2 protein in ALV-J non-permissible cells could induce susceptibility to ALV-J infection, the replication of ALV-J in the 293T cells transfected with chANXA2 was analysed by IFA and real-time PCR. Real-time PCR revealed that the relative expression of the ALV-J envelope gene was increased in the chANXA2-transfected 293T cells at 72 h post-infection . HoweverViral infection and pathogenesis are initiated by the binding of the viral surface protein to its cellular receptor. And the identification of viral cell receptor is critical not only for better understanding the molecular events for viral infection, but also for developing novel anti-viral strategies. The traditional methods for identifying viral receptors mainly depend on the expression and purification of the viral binding proteins111213The blocking of chANXA2 with a specific antibody and siRNA against chANXA2 can both effectively inhibit the infection/replication of ALV-J suggests chANXA2 can be as a novel host marker for anti-ALV-J strategies and as a new model system for examining the molecular events of retroviral entry into the host. To our knowledge, this is the first demonstration of the inhibition of ALV-J infection by antibodies against one of the host's protein, which highlights the fact that chANXA2 can serve as a novel functional receptor for ALV-J entry and provide a host marker for anti-ALV-J strategies. In 2006, chNHE1 was identified by Ning et al as a receptor for ALV-J infectionANXA2 is a member of the annexin family and plays vital roles in regulating cellular functions including the endo- and exocytotic pathways, the generation of plasmin and calcium-dependent F-actin filament bundling2022232527282950 by the Reed and Muench methodHEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Geese embryo fibroblast (GEF), DF-1 and pcDNA-env_DF1 cellsThe gp85 coding region including the signal peptide was amplified by PCR from the isolate JS09GY07. And the IgGFc heavy chain of rabbit was obtained with RT-PCR from the RNA of rabbit blood cells. The SU fragment and the IgGFc heavy chain were ligated with BamHI restriction enzyme site and the fusion fragment was cloned into the eukaryotic expression vector pcDNA3.1 for sequencing. After that, we cloned SUJ-IgGFc into adenovirus vector for expressing fusion protein in adenovirus expression system according to the manufacture's instruction. As expected, a recombinant adenovirus plasmid pAD-SUJ-rIgGFc containing a 1.8-kilobase (kb) fragment which fused ALV-J SU gene and the Fc region of rabbit IgG was constructed, and was transfected into 293T cell to generate recombinant adenovirus rAd-SUJ-rIgGFc which expressed fusion protein SUJ-rIgGFc, and it was purified with protein A agarose for subsequent protein precipitation.pcDNA-env_DF1 cells and DF1 cells in 100 mm dishes were harvested by scraping with a rubber policeman and homogenised with NP-40 lysis buffer containing 25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol (pH7.4) and a protease inhibitor cocktail (Roche). Intact cells and nuclei in the resulting extract were sedimented by centrifugation at 4°C for 5 min at 6000 g. The membrane proteins in the supernatant were sedimented by an additional spin at 13200 g for 1 h at 4°C and resuspended in 1% NP-40 lysis buffer. All extracts were stored at −80°C until use.The membrane proteins from the pcDNA-env_DF1 cells were immunoprecipitated with the monoclonal antibody JE9, which is specific to ALV-J Env and Resin A (Thermo Scientific). The membrane proteins from the DF1 cells were precipitated with the purified protein SUJ-rIgGFc and protein A agarose . Precipitated proteins were separated by SDS-PAGE, stained with a Silver Stain Kit (Thermo Scientific) and analysed with mass spectrometry. In Co-immunoprecipitation (Co-IP), 293T cells were co-transfected with plasmid pcDNA3.1-EnvJ, and chANXA2 for 48 h, and then lysed in lysis buffer. The cell lysates were immunoprecipitated with mAb JE9 at 4°C overnight, and then incubated with protein A agarose for 1 hr. After three washes in lysis buffer, the lysates were analyzed using JE9 and antibody against chANXA2 in western blot.50 and western blot.The DF1 cells were pre-treated with different concentrations of anti-ANXA2  for 2 h and then the cells were infected with ALV-J or ALV-A at an MOI of 5. The cells were maintained in DMEM with 1% fetal bovine serum and different concentrations of antibodies. Normal goat-IgG was used as the isotype control antibody in the blocking assay. The replication of ALV-J in the treated cells was analysed with IFA, real-time PCR, TCIDThree siRNA  against chANXA2 were synthesized (Invitrogen). The sequences of these siRNA were listed in 50/cell based on the titer obtained from DF1 cells) for 2 h followed by three washes with PBS and treatment with acid glycine (pH 3.0) for 1 min. After further three washes with PBS, the 293T and GEF cells were maintained in DMEM with 1% FBS for 48 h. For the 293T cells, the replication of ALV-J was analysed with IFA and real-time PCR at 12 h, 24 h, 48 h and 72 h post-infection. For the GEF cells, the cells and supernatants were homogenised and inoculated into the DF1 cells, and viral replication in the DF1 cells was analysed with IFA with JE9 at day 7 post-inoculation.293T or geese embryo fibroblasts (GEF) cells were transfected with chANXA2 for 48 h. Then, the transfected cells were infected with ALV-J at an MOI of 5  according to the manufacturer's instructions. The RNA was reverse-transcribed using PrimerScript RT Reagent Kit (Takara) with primers that are specific to the ALV-J gp37 and ANXA2 gene according to the manufacturer's instruction. Chicken 18S and human β-actin were used as an internal control in this assay. The primers of Real-time PCR for ALV-J gp37, ANXA2 and chicken 18S and human β-actin were listed in Indirect immunofluorescence assays (IFAs) were performed on the 293T and DF1 cells. The monoclonal antibody JE9, which is specific to the Env of ALV-J, was used as the primary antibodyM.M., A.J.Q., K.Q. and J.Q.Y. conceived and designed the experiments; M.M. and L.W. performed the experiments; M.M., A.J.Q., K.Q., X.M.H., H.X.S. and J.Q.Y. analyzed the data; M.M., L.W., X.M.H., H.X.S. and J.Q.Y. contributed reagents/materials/analysis tools; M.M., A.J.Q., J.Q.Y. and K.Q. contributed to the writing of the manuscript. M.M., L.W. and J.Q.Y. prepared figures. All authors reviewed the manuscript.