However, whether exosome-mediated microRNA transfer plays any role in cell invasion remains poorly understood. Thus, the aim of this study was to identify the exosomal microRNAs involved in breast cancer invasion.Exosomes are 30-100 nm membrane vesicles of endocytic origin, mediating diverse biological functions including tumor cell invasion, cell-cell communication and antigen presentation through transfer of proteins, mRNAs and microRNAs. Recent evidence suggests that microRNAs can be released through ceramide-dependent secretory machinery regulated by neutral sphingomyelinase 2 (nSMase2) enzyme encoded by the The expression level of endogenous and exosomal miRNAs were examined by real time PCR and the expression level of target proteins were detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study its uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-10b was estimated by invasion assay.In this study, we demonstrate that microRNA carrying exosomes can be transferred among different cell lines through direct uptake. miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cancer cells or non-malignant breast cells; it is actively secreted into medium via exosomes. In particular, nSMase2 or ceramide promotes the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppresses this secretion. Moreover, upon uptake, miR-10b can suppress the protein level of its target genes such as HOXD10 and KLF4, indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could induce the invasion ability of non-malignant HMLE cells.Together, our results suggest that a set of specific microRNAs may play an important role in modulating tumor microenvironment through exosomes. Thus, a better understanding of this process may aid in the development of novel therapeutic agents.The online version of this article (doi:10.1186/1476-4598-13-256) contains supplementary material, which is available to authorized users. The biological functions in higher organisms are executed by intercellular communication through complex assemblies of cells, the action of which must be highly coordinated. This cell-cell communication is also a vital aspect for development of various diseases including cancer. It has been demonstrated that the process of cellular communication is achieved by autocrine, paracrine or direct cell to cell contact. There is increasing evidence suggesting that cells may also communicate via other mechanisms in addition to these known methods, including exchange of cellular fragments, membranes or specialized organelles like microvesicles which until recently has been regarded as cellular debris. The two major forms of vesicles are ectosomes and exosomes. Ectosomes emerge from cellular membrane while the exosomes are of endosomal origin.Exosomes are nanovesicles with diameter ranging from 30-120 nm; they are derived from endocytic compartments and are released by many cell types including cancer cells. Exosome biogenesis is a complex and controlled process that begins with the inward budding of cellular membrane to form an early endosome. After the endosome formation, cytoplasmic content is internalized into small vesicles, i.e., exosomes. When exosomes are present inside the endosome, the endosome forms a multivesicular body (MVB) which later on fuses with the cellular membrane to release the exosomes–6. Also,Although it becomes evident now that microRNAs can be carried in exosomes and transported in the circulating system, it remains largely unknown whether these microRNAs have any function. In this study, we present evidence that exosomes can be transferred among different cell lines through direct uptake and in process delivers the microRNAs to the recipient cells. Upon uptake, microRNAs can still reduce the protein level of its target genes, indicating its functional significance. We found that miR-10b derived from MDA-MB-231 cells can be transferred to non-malignant HMLE cells via exosomes. Moreover, this transferred miR-10b can promote invasion in HMLE cells in part by targeting a known miR-10b target, HOXD10.HMLE cells were a generous gift from Dr. Robert Weinberg (The Massachusetts Institute of Technology). Primary antibodies were obtained as follows: monoclonal anti-β-actin and anti-HOXD10 from Sigma-Aldrich ; anti-KLF4 from Cell Signaling Technology . Secondary antibodies conjugated with IRDye 800CW or IRDye 680 were purchased from LI-COR Biosciences . Luciferase reporters, pMIR-D10UTR for HOXD10 3′UTR (#19117) and HOXD10 mut 3′ UTR (#19116), were purchased from Addgene (Cambridge MA). PCR primers and FAM-miR-10b were purchased from IDT .2 in a humidified chamber. For experiments, cells were grown in medium with exosome-free serum. Therefore, the serum added to the cell culture medium was depleted of exosomes by ultracentrifugation at 120,000 × g overnight (16 h) at 4°C, followed by passing it through 0.2 micron filter prior to use.The human MCF-7 and MDA-MB-231 cells were obtained from ATCC  and cultured in RPMI-1640 with 10% FBS . MCF-10A and HMLE cells were cultured in DMEM/F12K supplemented with growth factor hEGF, hydrocortisone, insulin and 100 units of penicillin/ml and 100 mg of streptomycin/ml. HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Cambrex) supplemented with 10% FBS. All media contained 2 mM glutamine, 100 units of penicillin/ml and 100 mg of streptomycin/ml. Cells were incubated at 37°C and supplemented with 5% COCells for exosome isolation from culture medium were maintained in respective medium with exosome-free FBS which was prepared by centrifugations to remove existing exosomes. Exosomes were isolated from cell culture medium collected after 48 h by differential centrifugations as described by Raposo and colleagues with a small modification. BrieflyThe high fidelity enzyme Phusion was used to amplify respective DNA fragments by PCR to make these constructs. To construct the GFP-CD63 fusion, we first amplified copGFP with primers copGFP-R1-5.1 and copGFP-CD63-3.1; CD63 with primers copGFP-CD63-5.1 and CD63-pCDH-PU-Not1-3.1. These two fragments were then cloned into pCDH-PU (SBI) at EcoR I and Not I site by cold fusion method (SBI). To clone miR-10b, a genomic DNA fragment (~500 bp) carrying miR-10b precursor was amplified from human genomic DNA with miR-10b-5.1 and miR-10b-3.1. The PCR fragment was first cloned into PCR8 (Life Technology) and then this fragment was migrated into pCDH-CMV-copGFP (SBI). SMPD3 expression vector was cloned by the same PCR amplification approach using primers SMPD3-R1-5.1 and SMPD3-Not1-3.1 and then cloned into pCDH-CMV-myc-EF1-GFP-PU at EcoR I and Not I site by cold fusion method. For cloning the KLF4 plus UTR, we used primers KLF4-Myc-R1-5.1 and KLF4-UTR-Not1-3.1, and then cloned into the pCDH-PU (SBI) at EcoR I and Not I restriction site. The sequences of all PCR products were verified by DNA sequencing.Cells were transfected with miR-10b mimic using RNAfectin reagent or with plasmid DNA using DNAfectin  following the manufacturer’s protocol.Confocal microscopy was carried out by using the Leica confocal microscope at the imaging facility of Southern Illinois University School of Medicine or Nikon C1 confocal microscope at Imaging Core at Center for Psychiatric Neuroscience of UMMC.2O, the samples were stained with 2% methylamine tungstate for 45 s and air-dried. Finally the samples were observed in a Zeiss scanning electron microscope.An aliquot of exosome suspension was loaded on to a carbon-coated electron microscopy grid. The sample was fixed with 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer at pH 7.3. After two washes in distilled HFor RT-PCR, we isolated total RNA using Trizol reagent  as per the manufacturer’s protocol and used 0.5 μg RNA to synthesize cDNA by SuperScriptase III (Invitrogen) with random primers. Finally, the resultant cDNA was used for PCR reactions. PCR annealing temperature varied depending on the primers used. To detect miR-10b, we used the polymerase A method, followed by SYBR Green qPCR as described previously. RNU1 anCells were harvested and protein was extracted from cells as previously described. The proTo determine whether miR-10b can confer invasion ability through exosome transfer, we overexpressed miR-10b in MDA-MB-231 cells and then isolated exosomes from the conditioned medium. The isolated exosomes were mixed with HMLE cells and incubated for 24 h before subject to invasion assays. Specifically, matrigel chambers (BD Biosciences) were used to determine the invasiveness per the manufacturer’s protocol as described previously. In brieLuciferase assays were carried out in HMLE cells. Cells were transfected with firefly luciferase reporters (pMIR-D10UTR for HOXD10 3′UTR and HOXD10 mut 3′ UTR) (400 ng) and 2 ng of the pRL-SV40 renilla luciferase construct in 12-well plates. Then they were incubated with appropriate exosomes for 24 h and finally harvested and lysed for luciferase assays. Luciferase assays were performed using a luciferase assay kit (Promega) according to the manufacturer’s protocol. Renilla luciferase was used for normalization.8. For infection, exponentially growing cells were mixed with viral particles in the presence of polybrene (0.8 mg/ml) in a six-well plate at a multiplicity of infection of 1-3.Lentiviral packaging was carried out in HEK-293T cells using a packaging system from System Biosciences as per the manufacturer’s protocol, as described previously. For a lSMPD3-R1-5.1 CCATGGAGGCCCGAATTCTGGTTTTGTACACGACCCCCSMPD3-Not1-3.1 TCGCAGATCCTTGCGGCCGCTCCGGACGGTCTATGCCTCCmiR-10b-5.1 GGTTAATAAAGCCGCCATCCmiR-10b-3.1 CTTCTGGAGAGGAAAAGCTGcopGFP-R1-5.1 ATTCTAGAGCTAGCGAATTCGCTAGACGCCACCATGGAGAcopGFP-CD63-5.1 CCGGCTCCACCGGATCTCGCGCGGTGGAAGGAGGAATGAAcopGFP-CD63-3.1 TTCATTCCTCCTTCCACCGCGCGAGATCCGGTGGAGCCGGKLF4-Myc-R1-5.1 CCATGGAGGCCCGAATTCTGAGGCAGCCACCTGGCGAGKLF4-UTR-Not1-3.1 TCGCAGATCCTTGCGGCCGCTTAATTCTCACCTTGAGTATGt-test was used for assessing the difference between individual groups and P ≤ 0.05 was considered statistically significant.Data are presented as mean ± SE; the Student’s Exosomes can be actively released from a variety of cell types including cancer cells. To determine whether microRNA-carrying exosomes can be released from breast cancer cells, we incubated three cell lines, i.e., MCF-10A, MCF-7 and MDA-MB-231, in exosome-free medium made from exosome-free FBS. The exosomes in the conditioned media were isolated by a serial centrifugations and filtration. The harvested exosomes were then resuspended in PBS or other agents depending upon further use. To deteTo examine whether the secreted exosomes can be taken up by recipient cells, we used two pronged approaches. In the first approach, we cloned CD63 along with GFP as a fusion protein in a lentiviral vector and then generated stable cell lines expressing CD63-GFP fusion. The distribution of CD63-GFP was mainly in speckle like structures Figure C, very dHaving demonstrated that exosomes are released from breast cancer cells and then taken by various types of cells, we determined what kind of microRNAs are secreted via exosomes. We chose metastatic MDA-MB-231 and non-malignant HMLE cells for comparison. We profiled the cancer-related microRNAs secreted in the conditioned media as well as the endogenous level in these two cell lines by qRT-PCR  machinery and is regulated by the sphingolipid ceramide[smpd3 gene coding for nSMase2 in a lentiviral vector. qRT-PCR analysis revealed that the level of secreted miR-10b was significantly higher in both MCF-7 and MDA-MB-231 cells overexpressing smpd3 than in vector control cells Additional file 1:


A recombinant plasmid targeting the Aurora-B gene (MiR-Aurora-B) was used to inhibit Aurora-B expression in HepG2 cells. Cell migration and invasion were investigated using Transwell migration and invasion assays. The results demonstrated that cell invasion and migration were suppressed by inhibiting Aurora-B. In addition, the effect of Aurora-B inhibition on the activity of the phosphoinositide 3-kinase (PI3K)/Akt/nuclear factor (NF)-κB signaling pathway was investigated by analyzing the protein expression levels of phosphorylated (p)-Akt, Akt, NF-κB p65, matrix metalloproteinase (MMP)-2 and MMP-9 using western blot analysis. The results demonstrated that the protein expression levels of p-Akt, NF-κB p65, MMP-2 and MMP-9 were reduced significantly by inhibiting Aurora-B. Therefore, inhibition of Aurora-B was shown to suppress hepatocellular carcinoma cell migration and invasion by decreasing the activity of the PI3K/Akt/NF-κB signaling pathway in vitro.In the present study, the effect of Aurora-B inhibition on HepG2 cell invasion and migration  Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide ,2. Althoin vitro and in vivo  are involved in the degradation of the basement membrane and epimatrix, among which MMP-2 and -9 markedly correlate with tumor invasion. A number of studies have revealed that activation of the gene encoding nuclear factor (NF)-κB, the upstream regulator of MMPs, promotes tumor cell invasion and migration ,13. In ain vitro.Thus, the aim of the present study was to investigate whether the inhibition of Aurora-B caused the suppression of HepG2 cell invasion and migration via decreasing the activity of the PI3K/Akt/NF-κB signaling pathway A human cDNA sequence encoding the Aurora-B protein was obtained from GenBank (NM-004217), and single-stranded DNA oligos were designed and synthesized using the following primer sequences: Forward, 5′-CCGGGAAGAGCTGCACATTTGACGACTCGAGTCGTCAAATGTGCAGCTCTTCTTTTTG-3′ and reverse, 5′-AATTCAAAAAGAAGAGCTGCACATTTGACGACTCGAGTCGTCAAATGTGCAGCTCTTC-3′.The products were cloned into the express vector, pcDNA6.2-GW/EmGFP-miR, using a BLOCK-iT™ Pol II miR RNAi Expression Vector kit with EmGFP . The DNA sequence of the plasmid was confirmed using a PureLink HiPure Plasmid DNA kit .2. The HepG2 cells were seeded in six-well plates at 30% confluence on the day prior to transfection. Transfection with the recombinant plasmid targeting the Aurora-B gene (MiR-Aurora-B) or the negative plasmid (MiR-Neg) was performed using Lipofectamine 2000 (Invitrogen Life Technologies). Transfection complexes were prepared according to the manufacturer’s instructions.A human HCC cell line, HepG2,  was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C in 5% COThe qPCR procedure was performed as follows: 95°C for 2 min; 35 cycles of 95°C for 20 sec, 55°C for 30 sec and 72°C for 30 sec; and annealing between 65 and 95°C with 0.5°C progressive increases. The primers used in the study were as follows: Aurora-B (NM-004217) forward, 5′-ATAGCAGTGGGACACCCGACAT-3′ and reverse, 5′-GGGACTTGAAGAGGACCTTGAGC-3′; and glyceraldehyde 3-phosphate dehydrogenase  forward, 5′-CCTGTTCGACAGTCAGCCGCATC-3′ and reverse, 5′-CGACCAAATCCGTTGACTCCGACC-3′.Total protein from the cells was extracted using radioimmunoprecipitation assay lysis buffer containing 60 μg/ml phenylmethylsulfonyl fluoride. Protein concentration was determined using a Bradford assay. Equal amounts of protein were electrophoresed using 8% SDS-PAGE and transferred onto a pure nitrocellulose blotting membrane (0.22 μM). The membranes were blocked with 5% skimmed milk for 1 h at room temperature, then incubated with primary antibodies  overnight at 4°C. The membranes were washed prior to incubation with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies . The immune complexes were detected with a pro-light HRP kit . GAPDH  protein expression was used as a normalization control for protein loading. All the experiments were repeated six times over multiple days.4 cells/well and cultured in DMEM with 2% serum, while 600 μl FBS-DMEM (10%) was added to the lower chamber. After 24 h, the migrated cells were fixed with 100% methanol for 30 min. The non-migrated cells were removed with cotton swabs. Next, the cells on the bottom surface of the membrane were stained with crystal violet for 20 min. Cell images were obtained under a phase-contrast microscope , and cell counts were performed using Image J software . Cell migration was assessed by determining the ability of the cells to move into a cellular space in a two-dimensional in vitro wound healing assay’ In brief, cells were grown to confluence in 6-well tissue culture plastic dishes to a density of ~5 × 106 cells/well. Cells were detached by dragging a rubber policeman  through the center of the plate. Cultures were rinsed with PBS and replaced with fresh DMEM or DMEM containing 10% FBS, after which the cells were incubated at 37°C for 24 h. Images were captured at 0 and 24 h and the migrated distance was measured using ImageJ . Transwell invasion and migration assays were repeated six times over multiple days.Cell invasion was assayed using a Transwell chamber  with Matrigel . For the invasion assay, a Transwell chamber was placed in a six-well plate and coated with 30 μl Matrigel, which was incubated for 40 min at 37°C. In the two Transwell assays, 24 h following transfection, the cells were trypsinized and seeded in chambers at a density of 5×10All measurement data are presented as the mean ± standard deviation. Statistical analysis was performed using a t-test for two independent-samples, where P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using SPSS version 13.0 .Cultured HepG2 cells were transfected with the recombinant plasmid for 6 h and then cultured for 48 h. Aurora-B mRNA and protein expression levels in the HepG2 cells were detected by qPCR and western blot analysis, respectively. Aurora-B mRNA and protein expression levels in the cells transfected with the recombinant plasmid were significantly lower compared with those transfected with the negative control plasmid . These oin vitro.HepG2 cells were transfected with MiR-Aurora-B or MiR-Neg. Transwell invasion and wound healing assays were performed to investigate the migration and invasion of HepG2 cells. The results revealed that the rates of invasion and migration in the cells transfected with MiR-Aurora-B were significantly lower compared with those transfected with MiR-Neg . These rTo investigate the effect of Aurora-B inhibition on the phosphorylation of Akt, the protein expression level of p-Akt was analyzed in HepG2 cells using western blot analysis. The results demonstrated that the p-Akt protein expression level in the cells transfected with MiR-Aurora-B was significantly lower compared with the cells that had been transfected with MiR-Neg . This obin vitro.Aurora kinases are serine/threonine kinases that are essential for cell cycle control and mitosis. Mammals have three Aurora kinase family members , and these kinases are expressed at maximum levels during mitosis. Aurora-B, part of the CPC, is located on the chromosome arms during prophase and at the centromeres during prometaphase and metaphase. Aurora-B subsequently localizes to the midbody during cytokinesis. Increased expression levels of Aurora-B have been identified in numerous types of cancer –17. Variet al demonstrated that inhibition of Aurora-B using a small molecular inhibitor was able to decrease the phosphorylation of Akt at Ser 473 (In addition, potential molecular mechanisms associated with Aurora-B inhibition of HCC cell migration and invasion were analyzed. The PI3K/Akt/NF-κB signaling pathway is known to be important in the metastasis of malignant tumors . Long et Ser 473 . Phospho Ser 473 , while t Ser 473 . During in vitro. Thus, inhibitors targeting Aurora-B and the PI3K/Akt/NF-κB pathway may be potential therapeutic strategies for treating HCC metastases.In conclusion, the observations of the present study indicate that inhibition of Aurora-B suppresses HepG2 cell invasion and migration via modulation of the PI3K/Akt/NF-κB signaling pathway


IL-17 is a pro-inflammatory mediator that is believed to play a critical role in regulating tissue inflammation during asthma, COPD, as well as other inflammatory disorders. The level of expression of IL-17 has been shown to be upregulated in lung bronchial tissue of asthmatic patients. Several reports have provided further evidence that this cytokine could play a key role in enhancing the migration of inflammatory as well as structural cells of the bronchial lung tissue during asthma and COPD. B cell infiltration to sites of inflammation during inflammatory disorders such as bowel disease, asthma and COPD has been reported. Accordingly, in this study we hypothesized that IL-17 may exert a chemotactic effect on primary B cells during asthma. We observed that B cells from asthmatic patients expressed significantly higher levels of IL-17RA and IL-17RC, compared to those of healthy subjects. Using an in-vitro migration assay, B cells were shown to migrate towards both IL-17A and IL-17F. Interestingly, blocking IL-17A and IL-17F signaling using either anti-IL-17R antibodies or MAP kinase inhibitors prevented in vitro migration of B cell towards IL-17. These observations indicate a direct chemotactic effect of IL-17 cytokines on primary peripheral blood B cells with higher effect being on asthmatic B cells. These findings revealed a key role for IL-17 in enhancing the migration of B cells to the lung tissue during asthma or COPD. Th-17 cells and their characteristic cytokines IL-17A, IL-17F, IL-21 and IL-22 play a beneficial role in the host-defense response against extracellular bacterial and fungal pathogens. However, they are also major harmful promoters in the pathogenesis of many chronic autoimmune and allergic disorders, including inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, and allergic asthma IL-17A and IL-17F cytokine signaling is mediated by specific receptors composed of IL-17RA and IL-17RC subunits, which are expressed on the cell surface of many cell types, including airway epithelial, airway smooth muscle and microvascular airway endothelial cells For many years, B cells were traditionally associated mostly with the production of antibodies. However, recent investigations have underlined their importance as modulators of the immune response in many chronic allergic and autoimmune diseases, through the release of cytokines and other inflammatory mediators. For example, B cells are important as a source of many regulatory cytokines such as TNF-alpha, lymphotoxin, IL-2, IL-6, IL-10, IL-13 and IL-17E among others Ten severe asthmatic patients  were recruited for this study. Refractory asthma classification was according to ATS criteria (ATS 1987) Peripheral venous blood (20 ml) was drawn from patients and control subjects. Blood was layered over Ficoll gradient  and centrifuged at 1000 g for 30 minutes. The mononuclear cells layer (PBMC) was then collected and B cells were isolated by negative selection using EasySep Human B cell enrichment kit . B cells purity was consistently >98% as evaluated by FACS analysis and the viability of freshly isolated B cells was >99% as evaluated by trypan blue dye exclusion. All experiments were performed using B cells isolated from the same 10 asthmatics and 10 control subjects.6 B cells were 19.2±2.6 µg for asthmatic eosinophils and 15.9±2.1 µg for healthy controls) . Levels of expression of IL-17RA and IL-17RC mRNA were then determined using quantitative RT-PCR . Relative expressions of IL-17RA and IL-17RC genes normalized with GAPDH were determined by the delta-delta Ct method B cells were stimulated with cytokines Th17 (50 ng/ml)) for 12 hours prior to cell harvest. Cells were then harvested, total RNA extracted  were stimulated or not with Th-17 cytokines for 24 hrs and then stained for IL-17RA and IL-17RC: cells were incubated for 1 hour on ice with the primary antibodies  conjugated with phycoerythrin (R&D Systems). Cells were washed using 1xPBS and fixed in 2% paraformaldehyde and analyzed by flow cytometry . Results were analyzed using DIVA software (BD Biosciences) and are presented as a percentage of positive cells.Isolated B cells (5×102/chamber). B cells were added to the upper chamber (50×103 cells), and different concentrations  of cytokines , or medium alone were added to the lower chamber. A preliminary experiment was performed to establish the time course of migration . Because the maximal increase in migration was observed at 18 hours (data not shown), all subsequent experiments were performed for 18 hours. After 18 hours of incubation at 37°C, the membrane was removed, and its upper face was scraped clear of cells. Cells that migrated to the lower side of the membrane were fixed and stained with the Hema 3 kit , according to the manufacturer’s instructions. The number of cells was counted in 5 random fields at a magnification x3200. Since B cells are non-adherent cells, migrating cells were also counted in the lower chamber by flow cytometry, and the total number of cells from both at the membrane and in lower chamber medium was obtained.B cells migration assays were performed as previously described To confirm that the increased migration associated with administration of exogenous Th17 cytokines was dependent on receptor activation, we pretreated B cells with anti–IL-17RA or anti–IL17RC mAb (R&D Systems) prior to migration assay. Results were compared with the corresponding Ig isotype controls.To investigate whether the chemotactic effects of exogenous Th17 cytokines could be mediated indirectly by stimulating the release of chemokines from B cells, we added either mouse anti-human CXCL8 (IL-8) (10 µg/ml) or anti-human CXCL-13 (0.3 µg/ml) monoclonal antibodies (R&D Systems) into both the upper and lower chambers during the migration, to block the potential effects of IL-8 and CXCL-13. In each case, results were compared with the effects of adding the corresponding Ig isotype controls. In an alternative approach, asthmatic B cells were stimulated with IL-17A, F or A+F cytokines (50 ng/ml) for 18 hrs. Supernatants were then collected and incubated or not with anti-IL-17A, F, or A+F (R&D Systems) neutralizing antibodies for 2 hr. The ability of these supernatants to enhance the migration of asthmatic B cells was then tested using Boyden chamber.3) were incubated with the inhibitors for 1 hour at 37°C prior to migration assay. Specifically, we used the p38 MAPK inhibitor SB203580 , the extracellular signal-regulated kinase (ERK) 1/2 MAPK inhibitor PD184352 , the NF-kB inhibitor PS1145 , and the phosphoinositide 3-kinase (PI3K) inhibitor PI103 . All results were compared with the corresponding vehicle control dimethyl sulfoxide.To determine the pathways involved in IL-17 induced B cell migration, we used pharmacologic inhibitors directed against various pathways that are potentially known to be activated by IL-17 and may be involved in cells migration. B cells . Protein lysates (10 µg) were then resolved on 10% acrylamide SDS-PAGE gel and blots were probed with antibodies to phosphorylated p38 MAPK (Millipore) and total p38 MAPK (Millipore). Membranes were analyzed with an Odyssey IR scanner using Odyssey imaging software 3.0 .2×10Statistical analysis for cytokine induced migration was first performed within the subgroups by using ANOVA and, when significant, followed by the post hoc Bonferroni test. The paired t test was performed for all other assays. P value of <0.05 was considered statistically significant. All data are presented as means ± SD.IL-17 cytokines were recently shown to be upregulated in airway tissue during asthma and to play critical roles in airway tissue remodeling. This includes enhancing the migration of structural and inflammatory cells such as smooth muscle cells, and neutrophils We next examined the ability of IL-17 cytokines to directly induce the migration of B cells in vitro using Boyden chamber migration assay. As shown in To confirm the requirement for activation of IL-17 pathway for B cell migration, we blocked IL-17R signaling on B cells using blocking anti-IL-17RA and anti-IL-17RC antibodies for 2 hrs prior to the chemotaxis assay with IL-17 cytokines. As shown in IL-17 could induce B cell migration indirectly by triggering the release of B cell chemoattractants from B cells. To investigate this possibility, we determined the migration of asthmatic B cells towards IL-17 in the presence of blocking antibodies to two known B cells chemoattractants, IL-8 and CXCL-13. As shown in We next explored the signaling pathways involved in the Th17 cytokine–induced migration of B cells. Using pharmacologic inhibitors, we tested for the involvement of p38 MAPK, ERK1/2 MAPK, NF-kB, and PI3K pathways in the chemotactic effect of Th17 cytokines. An inhibitor of p38MAPK, SB203580, partially inhibited the migration of B cells induced by IL-17A , IL-17F  and IL-17A+F  . In contAmple evidence supports a major role for Th-17-derived IL-17A and IL-17F cytokines in promoting chemotaxis indirectly, via upregulation of a large number of chemokines, including CXCL8 (IL-8) and CXCL13, which are involved in neutrophil infiltration in sub-epithelial airway tissues of severe asthmatics, and T and B lymphocyte migration in follicular germinal centers, respectively This is the first report, to our knowledge, unambiguously indicating that Th17 cytokines directly exert chemotaxis of B cells in vitro. IL-17A and IL-17F led to enhanced migration of human B cells in a mechanism that involved direct signaling via IL-17R and activation of p38 MAP kinase. In the context of the growing recognition of the critical involvement of B cells in asthma pathogenesis, not only via the production of IgE, but of various regulatory mediators, our findings underscore the key role Th17-associated cytokines play in airway tissue remodeling and asthma pathogenesis.Th17 cells are involved in a variety of human diseases, particularly in allergic and chronic autoimmune diseases In this study, we first confirmed the presence of Th17 cytokine receptors on B cells isolated from asthmatic and healthy recruited patients . InteresIL-17A and IL-17F cytokines promoted chemotaxis of B cells in vitro . These eIL-17A and IL-17F homodimers have both been shown to bind independently to IL-17RA and IL-17RC To explore the molecular mechanism underlying the chemotactic effect of Th17 cytokines on B cells, we tested the involvement of various MAP kinase pathways known to be activated by IL-17R triggering. Of all the MAPK inhibitors used, only SB203580, which inhibits p38α and p38β MAPK isoforms significantly inhibited B cell migration. Activation of p38 MAPK by IL-17A/F was further confirmed by the assessment of p38 MAPK phosphorylation. IL-17A and IL-17F exhibited similar kinetics for the activation of p38 MAPK, suggesting that they fulfill similar biological functions using the same signaling pathways. To our knowledge, this is the first demonstration of p38 MAPK activation by IL-17 in B cells, which is congruous with previous observations in microvascular airway endothelial cells, indicating that activation by IL-17 occurs in a p38 MAPK-dependent manner to upregulate endothelial adhesion markers , and to improve IL-8 mRNA stability in smooth muscle cells In summary, we have demonstrated for the first time that IL-17A and IL-17F play a key role in inducing B cell migration. This novel observation, together with published evidence of chemotaxis activity by IL-17 on airway smooth muscle cells


HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase gene, e.g. positions L74I, S153A, G163Q and T206S. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on susceptibility to integrase inhibitors and emergence of resistance associated mutations.We cloned and purified integrase proteins from each of HIV-1 Group O clades A (HIV-O/A) and B (HIV-O/B), a HIV-O divergent strain (HIV-O/Div), and HIV-1 group M  and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of varying concentrations of several INSTIs. The inhibition constant (Ki) and IC50 were calculated and compared for HIV-M and HIV-O integrases. Selections for resistance-related mutations were performed using cord blood mononuclear cells and increasing concentration of INSTIs.HIV-O integrase and viruses were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. During selection, we observed different pathways of resistance depending on the drug and clade. Mutations selected in HIV-O can be classified as follows: (1) mutations described for HIV-M such as T97A, Q148R, V151A/I (RAL), T66I, E92Q, E157Q (EVG) and M50I, R263K (DTG) and (2) signature mutations for HIV-O (i.e. not described in HIV-M) F121C (HIV-O/B for RAL), V75I (HIV-O/A for RAL) and S153V (HIV-O/A for DTG). Only the HIV-O/Div selected the Q148R mutation for RAL and R263K+M50I for DTG, as previously described for HIV-M. None of the HIV-O viruses selected either N155H or Y143C. The selection of the specific S153V mutation could be explained at the nucleotide level: HIV-O at this position contains an alanine and substitution of alanine to valine (153AGGC→153VGTC) is easier than substitution of alanine to tyrosine (153AGGC→153YTAC), with only a transversion needed instead of one transition plus one transversion.This is the first report of susceptibility and resistance in vitro to INSTIs for HIV-O. Our study confirmed the impact of HIV-O polymorphism, on susceptibility to INSTIs and the emergence of resistance mutations.


We demonstrated that Aire cells upregulated the mRNA levels of the tolerance-related molecules CD73, Lag3, and FR4 and the apoptosis of CD4+ T cells in STZ-T1D mouse-derived splenocytes. Furthermore, following insulin stimulation, Aire cells decreased the number of CD4+ IFN-γ+ T cells in both STZ-T1D and WT mouse-derived splenocytes and reduced the expression levels of TCR signaling molecules (Ca2+ and p-ERK) in CD4+ T cells. We observed that Aire cells-induced CD4+ T cells could delay the development of T1D. In summary, Aire-expressing DCs inhibited TCR signaling pathways and decreased the quantity of CD4+IFN-γ+ autoreactive T cells. These data suggest a mechanism for Aire in the maintenance of peripheral immune tolerance and provide a potential method to control autoimmunity by targeting Aire.Autoimmune regulator (Aire) can promote the ectopic expression of peripheral tissue-restricted antigens (TRAs) in thymic medullary epithelial cells (mTECs), which leads to the deletion of autoreactive T cells and consequently prevents autoimmune diseases. However, the functions of Aire in the periphery, such as in dendritic cells (DCs), remain unclear. This study’s aim was to investigate the effect of Aire-overexpressing DCs (Aire cells) on the functions of CD4 In the thymus, T cells develop, mature and form a polyclonal T cell repertoire capable of identifying large numbers of exogenous pathogens to prevent the occurrence of diseases. During this process, T cell clones that recognize self-antigens are inevitably generated. To maintain self-tolerance of a normal body, T-cell clones that react with self-antigens need to be depleted . AutoimmAire is mainly expressed in medullary thymic epithelial cells (mTECs) [Aire gene causes autoimmune polyglandular syndrome type I (APSI) [Among the central immune organs,  (mTECs) . Aire re (mTECs) ,10,11,12I (APSI) , which iI (APSI) ,15.Aire expression has also been observed in peripheral tissues, especially in peripheral blood and lymph node-derived DCs, macrophages, and epithelial cells. However, the role of Aire in peripheral tissues is poorly understood [in vitro, macrophages overexpressing Aire elevated a subset of CD4+Foxp3+ Treg cells [Aire are capable of delaying the occurrence of MOG-induced experimental autoimmune encephalomyelitis (EAE) [+ DCs in the spleen [Recently, derstood ,16,17,18eg cells ; howevereg cells . Bone mais (EAE) . Moreovee spleen . A studye spleen ,26,27,28+ T cells. Furthermore, the mechanism by which Aire-overexpressing DCs induced the functional inactivation of CD4+ T cells was explored. Finally, the effects of CD4+ T cells induced by Aire-overexpressing DCs on the incidence of T1D in mice were examined. The results showed that Aire induced tolerance in T1D-related autoreactive T cells and prevented the occurrence of T1D by regulating the expression of cell surface molecules and T1D-associated TRAs in DCs.In the present study, we utilized the Aire-overexpressing DC cell line DC2.4 to examine the effect of Aire on molecules related to DC tolerance. Based on these findings, the Aire-overexpressing DC cell line DC2.4 was co-cultured with splenocytes derived from mice with streptozotocin (STZ)-induced T1D to examine the effect of Aire-overexpressing DCs on the tolerant status of CD4II (MHC II) between the two groups of unstimulated cells (glutamic acid decarboxylase 65/67 (GAD65/67), insulin-2 (Ins2), insulinoma antigen 2(IA-2) and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) were significantly elevated in the Aire cells compared with the control cells. The expression of insulin-like growth factor 2 (IGF2) and chorionic gonadotropin alpha chain (CGA) was not detected in either the Aire or control cells  and wild type (WT) mice were co-cultured with either Aire or control cells for 48 h. Then, we examined CD4+ T cells apoptosis by flow cytometric analysis. The results showed that the apoptotic rate of STZ-T1D mouse-derived CD4+ T cells was higher in the Aire group compared to the control group. In contrast, no significant difference was detected in the apoptotic rates of WT mouse-derived CD4+ T cells between the two groups , folate receptor 4 (FR4) and programmed cell death protein 1 (PD-1), and the mRNA expression level of the cytokine interleukin 2 (IL-2) in T cells after co-culturing splenocytes and Aire cells [+ T cells, reduced the number of IFN-γ-producing autoreactive CD4+ T cells in STZ-T1D and WT mouse and upregulated the expression of tolerance-related molecules in splenocytes, suggesting that Aire-overexpressing DCs may play a role in maintaining the tolerant state of CD4+ T cells.To confirm the tolerant status of CD4re cells ,31. The + T cells derived from STZ-T1D mice by affecting TCR signaling pathways. STZ-T1D and WT mouse-derived splenocytes were co-cultured with either Aire cells or control cells and then stimulated with an anti-CD3 antibody, concanavalin A (ConA) or insulin. Subsequently, we examined the key molecules of the TCR signaling pathways in CD4+ T cells by fluorescence-activated cell sorting (FACS), including Ca2+ and phosphorylated extracellular signal-regulated kinase (p-ERK) [2+ and p-ERK levels after stimulation with the CD3 antibody and insulin were lower in the STZ-T1D and WT mouse-derived CD4+ T cells from the Aire group compared with those from the control group. Although the levels of Ca2+ and p-ERK were elevated in STZ-T1D and WT mouse-derived CD4+ T cells upon stimulation with the unrelated control ConA compared to the unstimulated CD4+T cells, there were no significant difference in the levels of Ca2+ and p-ERK between the Aire and control groups after ConA stimulation (2+ and p-ERK) in STZ-T1D mouse-derived CD4+ T cells, resulting in the functional inactivation of autoreactive CD4+ T cells. Thus, this may be one pathway by which Aire induces tolerance in autoreactive T cells.The T cell receptors (TCRs) located on the surface of the autoreactive T cells recognize the major histocompatibility complex class II (MHC II) complexes formed after DC-mediated antigen presentation. Antigen recognition activates the intracellular TCR signaling pathways and promotes the production of IFN-γ by activated T cells, resulting in autoimmune diseases . Therefo (p-ERK) . The resmulation . These r2+ and p-ERK were markedly reduced in CD4+ T cells after splenocytes derived from WT mice were co-cultured with Aire cells and stimulated with insulin. These results led us to speculate whether Aire-induced CD4+ T cells would inhibit or delay the occurrence of T1D in STZ-induced mice. In the following experiment, splenocytes derived from WT mice were co-cultured with Aire cells or control cells for 48h. The purity  expressing low levels of CD80 and CD86 may serve as tolerant cells to induce antigen-specific immune tolerance. Additionally, stimulation of Toll-like receptors (TLRs) failed to reverse eTAC-induced immune tolerance ,35,36. T+ DCs expressed a high level of Ins2 compared to the total mouse splenocytes [One study showed that DCs present in the periphery also expressed TRAs and played a role similar to mTECs  in whichenocytes . Therefoproduction of significant levels of IFN-γ by T cells is the main cause of autoimmune diseases such as T1D. The peripheral immune system maintains a state of tolerance by restricting IFN-γ+ T cells to a low percentage of total CD4+ T cells [+ IFN-γ+ T cells among the total splenocytes. These results indicated that Aire-overexpressing DCs played a role in preventing the occurrence of T1D by inhibiting the production of IFN-γ by CD4+ T cells or inducing the apoptosis of IFN-γ-producing autoreactive CD4+ T cells.Previous reports have shown that the  T cells ,40,41. I+ T cells recognize specific antigens presented by MHC II molecules through TCRs and then receive stimulatory signals from the engaged TCRs. In the presence of synergistic actions of costimulatory molecules, TCR signaling pathways will be fully activated and further promote the production and release of downstream cytokines, such as IFN-γ, thereby eliciting immune responses. Loss of costimulatory molecules leads to the inactivation of TCR signaling pathways [+ IFN-γ+ T cells in mouse splenocytes was caused by the inactivation of TCR signaling pathways in CD4+ T cells. In the present study, splenocytes derived from STZ-T1D and WT mice were co-cultured with Aire-overexpressing DCs and then stimulated with insulin. The results showed that the levels of key molecules of the TCR signaling pathways (Ca2+ and p-ERK) were significantly decreased in STZ-T1D and WT mouse-derived CD4+ T cells after induction with Aire-overexpressing DCs. These results suggest a mechanism in which Aire suppresses the activity of CD4+ T cells by inhibiting the expression of costimulatory molecules during the process of DC activation and inhibiting TCR signaling pathways in CD4+ T cells, eventually leading to a reduction in the number of CD4+ IFN-γ+ T cells.CD4pathways . The res+ T cells that exhibit high affinity for the TRAs [+ T cell apoptosis in STZ-T1D and WT mouse-derived splenocytes after co-culturing with Aire-overexpressing DCs. Our results were consistent with previous reports in the literature that demonstrated that Aire-transfected DCs significantly enhanced the apoptosis of STZ-T1D mouse-derived CD4+ T cells, whereas they exerted no significant effect on WT mouse-derived CD4+ T cells. These results indicated that Aire induced the apoptosis of autoreactive CD4+ T cells by promoting the expression of TRAs in DCs, which may represent another mechanism for the maintenance of immune tolerance. However, one cannot rule out the possibility that Aire-expressing cells may induce CD4+ T cell tolerance through other means, such as direct cell-cell contact or soluble mediators. We have measured the level of some soluble mediators, such as TGF-β was upregulated in Aire overexpressing cells. This may contribute to this effect. We also tried to investigate the effect using transwell cocultures, but unexpectedly there was no change in the number of regulatory T cells. We therefore think this effect may also depend on direct cell-cell contact. We will further investigate the other mechanisms involved the effect of Aire overexpression in our future study. In summary, Aire-overexpressing DCs maintain CD4+ T cell tolerance by inducing the functional inactivation and apoptosis of autoreactive CD4+ T cells.The expression of TRA genes in the thymus provides an opportunity for developing T cells to contact peripheral self-antigens and induces the apoptosis and clearance of CD4the TRAs ,44,45. Tthe TRAs ,4. The r+ T cells using in vitro assays. To investigate whether CD4+ T cells were capable of resisting activation after the induction of tolerance and truly played a role in preventing the occurrence of T1D, splenocytes derived from WT mice were co-cultured with Aire-overexpressing DCs and then adoptively transferred into WT mice. Then, the mice were induced with STZ. The blood glucose levels were significantly reduced in mice adoptively transferred with Aire-overexpressing DC-induced splenocytes. Furthermore, H&E staining of mouse pancreatic tissues showed a low degree of inflammatory infiltration. The IFN-γ and p-EPK expression levels were markedly reduced in CD4+ T cells among the total splenocytes following insulin stimulation. These results indicated that CD4+ T cells induced with Aire-overexpressing DCs were capable of delaying the occurrence of T1D in STZ-induced mice.Other studies have reported that T1D is caused by autoreactive T cells. Once activated, autoreactive T cells enter into pancreatic tissues, form inflammatory infiltrates and produce large amounts of IFN-γ, which leads to the further destruction of pancreatic β cells. Eventually, pancreatic β cells lose the ability to secrete insulin ,41. In aC57BL/J6 mice bone marrow cultures followed by supertransfection with myc and raf oncogenes [The dedritic cell line DC2.4 was established by transducing granulocyte-macrophage CSF into ncogenes , obtainencogenes . C57BL/JC57BL/J6 mice and gently dissociated into single-cell suspensions. Aire cells or pEGFPC1 cells (2 × 105 per well) and splenocytes (4 × 106 per well) were seeded into six-well plates. Once the cells attached to the dishes, 5 mg/mL ConA , 10 mg/mL insulin  or 5 μg/mL anti-CD3 antibody  were added and the plates were incubated for 48 h. Finally, the splenocytes were harvested for flow cytometry and real-time PCR analyses.The spleens were removed from the −∆∆Ct. The following primers were used: GAPDH , Gad67 , IGF-2 , GAD65 , IA-2 , Ins2 , chrA , IGRP , Lag3 , FR4 , PD-1 , and IL-2 .Total RNA was extracted from the harvested splenocytes as described above or from the Aire and pEGFPC1 cells using RNAiso™ PLUS  and dissolved in DEPC-treated water. The amount of total RNA was measured using an ultraviolet spectrophotometer . cDNA was synthesized from 1.0 μg of total RNA using the M-MLV reverse transcriptase and oligo(dT) in a total volume of 20 μL according to the manufacturer’s instructions (Takara). Real-time quantitative PCR (qPCR) was performed using an ABI PRISM 7300 sequence detection system  with SYBR Premix Ex Taq™ II (Takara) following the manufacturer’s suggested protocol and using the following conditions: 95 °C for 30 s and 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The results were analyzed using the formula 26 cells were suspended in 100 µL of PBS. The cells were incubated with PE-anti-CD40, PE-anti-CD80, PE-anti-CD83, PE-anti-CD86, PE-anti-Cd11C, PE-anti-MHC II and PE-Cy7-anti-CD4  on ice for 45 min and subsequently fixed with fixation/permeabilization concentrate with diluent (eBioscience) for 1 h. Alternatively, the cells were incubated with Fluo-3 for 30 min at room temperature for Ca2+ staining. Then, the cells were treated with 0.1% saponin  and anti-IFN-γ-PE, anti-phosphorylated-ERK, and anti-IgG-PE  at 4°C for 1 h. Next, the cells were washed with PBS and resuspended in 2% paraformaldehyde for analysis using a BD FACS Calibur flow cytometer.The cells were collected and counted, and 1 × 10C57BL/6J mice were treated by intraperitoneal (i.p.) injection of STZ (80 mg/kg) dissolved in cold 0.1 M sodium citrate buffer solution once a day for five consecutive days . The ons6 splenocytes were intravenously (i.v.) injected into the tail veins of 8-week-old mice, once every 3 days and repeated for three times. Two days after the first transfer of splenocytes, the mice were induced by STZ as described above and observed for the onset of diabetes by measuring blood glucose levels.After co-culture with the DC2.4 cell line as described above, 2 × 10The pancreas was removed from mice and fixed in formalin, embedded in paraffin, sectioned, and stained with H & E.t-test. The statistical significance between two groups was set at p < 0.05.All experimental data are reported as the means, and the error bars represent the experimental standard errors. Statistical analysis was performed using Student’s + T cells. Our data suggest that peripheral CD4+ T cell tolerance can be maintained through the combined effects of the two mechanisms and indicate that approaches to modulate Aire expression in DCs could be a potential method to control autoimmunity.In conclusion, our study shows that Aire-overexpressing DCs play a role in inducing the functional inactivation and apoptosis of autoreactive CD4


Type 1 diabetes mellitus (T1D) accounts for ~10% of all diabetes cases, and it is caused by autoimmune destruction of pancreatic beta-cells, which leads to insulin deficiency and fates individuals to require insulin treatment to survive. The triggering of autoimmunity against beta-cells is caused by interaction between environmental and genetic risk factors. Among the several loci associated with T1D, the human leukocyte antigen (HLA) class II DR/DQ locus is the main genetic risk factor for T1D, accounting for 30-50% of genetic risk for this disease. Other genes have been associated with minor effects on T1D risk when compared with HLA, with different studies indicating that the effect of non-HLA polymorphisms on predisposition for T1D may be different according to HLA DR/DQ types. In this scenario, a recent study identified a minimum set of three polymorphisms  which can predict high-risk HLA-DR/DQ types relevant to T1D.To evaluate frequencies of high-risk T1D HLA-DR/DQ haplotypes in a Southern Brazilian population using a minimum set of HLA polymorphisms .We analyzed 387 T1D patients (cases) and 375 healthy blood-donor subjects (controls). The local ethics committee approved the protocol, and all patients signed an informed consent form. Polymorphisms of interest were genotyped by allelic discrimination – RT-PCR technique using TaqMan MGB probes (Life Technologies). Haplotype combinations of the three analyzed polymorphisms were used for defining the HLA types relevant to T1D , distinguishing the highest-risk DR4-DQ8 and DR3/4-DQ types.Minor alleles frequencies of rs3104413, rs2854275, rs9273363 were increased in T1D patients as compared to non-diabetic subjects .The frequency of high-risk DR4-DQ8 type was 66.7% in T1D cases and 15.3% in controls .The high-risk DR3/4-DQ8 heterozygous haplotype was observed in only one T1D patients and in none control subject.As expected, the high-risk HLA-DR4/DQ8 haplotype is associated with increased risk for T1D in our population. The genetic risk of non-HLA genes on T1D in Southern Brazil can now be correct for different high-risk HLA-DR/DQ types.


This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse  linear IgG1 and three rabbit  linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15–120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R Molecular weight markers that can be auto-detected by secondary antibodies and tolerate denaturing conditions can offer great convenience for protein analysis by Western Blotting. Currently, dye-conjugated molecular weight protein markers are commonly used in protein analysis , 2. HoweE.coli outer membrane protein intimin domain 2 , maltose-binding protein  or an E.coli transcription factor  to generate 15–120 kDa molecular weight marker proteins (M&R LE protein marker) (E. coli (BL21) was confirmed by anti-His staining and Western blotting. The auto-detecting nature of the M&R LE protein markers was examined by direct staining on immunoblots with anti-mouse and anti-rabbit secondary antibodies. The molecular weight accuracy of the M&R LE protein marker was also determined by calculating the correlation  of the relative mobilities versus the logarithmic molecular weights in comparison with a commercial prestained marker. The M&R LE protein marker can be directly recognized by secondary antibodies under denaturing conditions for automatic visualization on immunoblots. These new markers provide an accurate, convenient and effective tool for protein analysis and proteomic research.To develop an accurate, denaturable and auto-detecting molecular weight marker, we selected three mouse  and three rabbit  linear epitopes (LE) through prediction of the crystal structures of mouse IgG1 and rabbit IgG heavy chain constant regions by ABC prep, Bepi prep and C.V.C. software. We expected that the linear epitopes exposure at constant region of primary antibodies are potentially bound by secondary antibodies after structural destroyed by heating –17. We d marker) . The proSecondary antibodies that were used to directly stain immunoblots included: horseradish peroxidase (HRP)-labeled goat anti-mouse IgG Fc (115-035-006), HRP-goat anti-rabbit IgG (115-035-003), HRP-goat anti-mouse IgG, A, M (115-035-006), HRP-goat anti-mouse IgM (109-035-006) , HRP-goat anti-mouse IgG (H+L) (626520) , HRP-goat anti-mouse IgG (H+L) (31430) and HRP-goat anti-rabbit IgG (H+L) (31460) , and HRP-goat anti-rabbit IgG (H+L) (656120) . All secondary antibodies were diluted 1000-fold in phosphate buffered saline (PBS) with 5% milk for immunoblotting experiments.http://www.imtech.res.in/raghava/abcpred/), BepiPred 1.0 Server (http://www.cbs.dtu.dk/services/BepiPred/) and Cancer Vaccine Center (CVC) bioinformatics (http://bio.dfci.harvard.edu/research/bioinformatics-tools.php) were used to predict the linear epitopes in the antibody Fc regions. Three mouse  and three rabbit  linear epitope candidates were selected based on the predicted results. The M1 (TPKVTCVVVDISKDDPEVQF SWFVDDVEVHTAQTQPREEQFNSTFRSVSELP), M2 (EFKCRVNSAA FPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITD) and M3 (QPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGL) peptides were derived from the mouse IgG1 heavy chain CH2 and CH3 domains. The R1 (PKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRT ARPPLREQQFNSTIRVVSTLPIT), R2 (GKEFKCKVHNKALPAPIEKT ISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEW) and R3 (KNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHEANHYTQKSISRSPGK) peptides were derived from the rabbit IgG heavy chain CH2 and CH3 domains.The gene sequences of mouse IgG1 (PDB number: 3HKF) and rabbit IgG antibodies (PDB number: 2VUO) were obtained from the National Center of Biotechnology Information (NCBI) and the Protein Data Bank (PDB). ABCpred (Escherichia coli (E.coli) outer membrane protein intimin domain 2 , maltose-binding protein  and an E.coli transcription factor . Candidate DNA fragments of M1, M2, M3, R1, R2 and R3 peptides were assembled by PCR and cloned into the pRSETB plasmid (Invitrogen). The M2R2 fusion sequences were created by digesting the R2 sequence with BglII and PciI and subcloning the DNA fragment into the pRSETB-M2 plasmids to generate pRSETB-M2R2 plasmids (M&R epitope). A sequence coding for a His-tag (six histidine) was digested with NcoI and AvaI and inserted behind the M2R2 fusion sequence to generate pRSETB-M2R2-His tag (15 kDa). The pRSETB-M2R2-His tag (15 kDa) was digested with AvaI and the D2 DNA fragment was inserted to generate pRSETB-M2R2-D2-His tag (25 kDa). pRSETB-M2R2-His tag (15 kDa) was digested with NdeI and EcoRI and the MAS DNA fragment was inserted in front of the M2R2 sequence to generate pRSETB-MAS-M2R2-His tag (30 kDa). pRSETB-MAS-M2R2-His tag (30 kDa) was further digested with AvaI and ClaI and the D2 DNA fragment was inserted to create pRSETB-MAS-M2R2-D2-His tag (40 kDa). pRSETB-MAS-M2R2-D2-His tag (40 kDa) was digested with KpnI and EcoRI and another D2 DNA fragment was inserted to create pRSETB-MAS-M2R2-D2-D2-His tag (50 kDa). Consequently, pRSETB-MAS-M2R2-D2-D2-D2-His tag (60 kDa) was generated by PmlI and KpnI digestion of pRSETB-MAS-M2R2-D2-D2-His tag (50 kDa) and insertion of another D2 gene sequence. To construct pRSETB-MAS-M2R2-D2-MBP-His tag (80 kDa), pRSETB-MAS-M2R2-D2-His tag (40 kDa) was digested with ClaI and StuI, and then a fragment coding MBP was inserted behind the D2 sequence. pRSETB-MAS-M2R2-D2-Nus-His tag (100 kDa) was created by HindIII and EcoRI digestion of pRSETB-MAS-M2R2-D2-His tag (40 kDa) and insertion of a fragment coding the Nus sequence. Finally, the pRSETB-MAS-M2R2-D2-D2-D2-Nus-His tag (120 kDa) was generated by HindIII and ClaI digestion of pRSETB-MAS-M2R2-D2-D2-D2-His tag (60 kDa) and insertion of a Nus sequence.The polymerase chain reaction (PCR) was used to amplify genes including molecular adjusting sequence , O157 E. coli BL21 (DE3) (Invitrogen) cultured in Luria bertani (LB) broth containing 0.1 mg/mL ampicillin  at 37°C. When the culture absorbance at 600 nm reached 0.5, protein expression was induced by adding 0.2 mM isopropyl β-D-thiogalactopyranoside (IPTG)  at 37°C for 3 hours. The transformed BL21 cells were collected for Western blotting analysis. The components of the M&R LE protein marker were stored in the refrigerator at -20°C.The plasmids of the M&R LE marker proteins were expressed in 6 BL21 cells expressing M&R LE protein markers were harvested, mixed with sodium dodecyl sulfate (SDS) loading dye containing β-mercaptoethanol (Sigma-Aldrich) and boiled for 10 min. Five to twenty μL samples were electrophoresed on a 10% or 12.5% SDS-PAGE and then electrophoretically transferred to nitrocellulose (NC) membranes . The membranes were blocked in PBS containing 5% milk at room temperature for 2 hours. For analysis of protein expression, the NC membranes were stained with mouse anti-histidine (His) tag antibody  at room temperature for 1 hour. After washing 3-times with PBS containing 0.1% Tween-20 (Sigma), the NC membranes were incubated with goat anti-mouse IgG Fc-HRP (Jackson Immuno-Research Laboratories) at room temperature for 1 hour. After extensive washing, the blots were developed by ECL  and chemiluminescence was captured on a UVP BioImaging system . To test the auto-detected activity of the M&R LE protein marker, the marker proteins were transferred onto NC membranes and directly stained with goat anti-mouse IgG Fc-HRP, goat anti-rabbit IgG Fc-HRP (Jackson Immuno-Research Laboratories), goat anti-mouse IgG (H+L)-HRP (Invitrogen) or goat anti-rabbit IgG (H+L)-HRP (Thermo Scientific) secondary antibodies at room temperature for 1 hour and then detection was performed as described above.5 × 10To examine the relationship between molecular size and mobility of M&R LE marker proteins, the logarithm of the protein molecular weights (in kDa) was plotted against the relative mobility (Rf) of the M&R LE marker proteins on immunoblots. The data was fit by simple linear regression using SigmaPlot 10.0 software .E. coli. Bacterial lysates were resolved on a SDS-PAGE and immunoblotted with an anti-His tag antibody to detect the His epitope tag present in the C-terminus of the IgG Fc LE. All six IgG Fc LE peptides were expressed in BL-21 E. coli  for binding by commercial secondary antibodies. IgG Fc LE candidate sequences from mouse  primary IgG1 and rabbit  IgG antibodies were predicted based on their crystal structures by ABCpred, BepiPred 1.0 Server and Cancer Vaccine Center (CVC) bioinformatics software . We clon E. coli . To inve E. coli  or anti- E. coli  secondar E. coli  and HRP- E. coli , respect E. coli . A simil E. coli . These dE. coli. Immunoblotting with anti-His tag antibody showed that the M&R epitope was successfully expressed by BL-21 E.coli  to form a protein ladder with regular molecular weights , indicating that the M&R LE protein marker is highly accurate (2 = 0.9735), possibly owing to altered electrophoretic mobilities caused by dye molecules on the marker proteins. These results indicate that the M&R LE protein marker displays precise molecular weights as compared with a commercial prestained marker.The migration of the M&R LE protein markers in immunoblots was analyzed to determine the accuracy of the markers. The relative mobility of the component protein markers, which is defined as the ratio of the protein migration distance to the distance of the running front in the SDS-PAGE, was plotted against the logarithm of the protein molecular weight. Linear regression analysis demonstrated that the relative marker mobilities and the logarithm of the marker molecular weights were inversely correlated . The M&R LE protein marker is recognized by HRP-conjugated anti-mouse and anti-rabbit secondary antibodies under denaturing conditions and auto-detects a regular molecular weight ladder on immunoblot films. This novel strategy may provide a more accurate, convenient and effective tool for proteomic research.in situ hybridization [Antibodies are widely used in several types of immunoassays such as immunoblotting , 19, immdization , 25. Becdization , commercdization . Furtherdization . We therA protein ladder which can tolerate denaturing conditions is important to the development of molecular weight markers. Previous study had suggested that the linear epitope can successfully bind by antibodies after heating treatment (100°C) . Unlike The M&R LE protein marker can be widely applied in immunoblots that use anti-mouse or anti-rabbit IgG Fc secondary antibodies in immunoblot. However, it cannot be used with assays that employ anti-Fab, anti-IgA or anti-IgM secondary antibodies nor secondary antibodies against other species. In this study, we provide a novel strategy based on the identification of linear epitopes in the primary antibody that can be used as a detectable tag for secondary antibodies. This concept should be extendable to selection of linear epitopes from different portions of antibodies such as the Fab region or to fusing linear epitopes of primary antibody from two or more different species to generate second-generation auto-detected protein markers.In summary, we developed a novel M&R LE protein marker for protein electrophoresis. It has apparent advantages over traditional protein markers as follows: (1) the marker can be recognized by secondary anti-mouse and anti-rabbit antibodies under reducing conditions, (2) the marker displays precise molecular weights, (3) the marker can be auto-detected on film in immunoblots, (4) it is suitable for any anti-mouse and anti-rabbit IgG Fc secondary antibodies and (5) can act as a positive control for secondary antibodies. We expect that the M&R LE protein marker will provide a denaturable, accurate and convenient standard for protein electrophoresis.S1 FigSerially-diluted M&R LE protein markers were directly stained by HRP-conjugated (A) anti-mouse or (B) anti-rabbit IgG Fc secondary antibody at concentration of 0.8–1.6 μg/ml. The molecular weights of a commercial pre-stained protein marker (right) and the M&R LE protein markers (left) are indicated.(TIF)Click here for additional data file.


Wilms Tumor 1 (WT1) mutations and variants are implicated in several diseases, including Wilms tumor and acute myeloid leukemia (AML). We observed two alternative G-to-A changes, namely c.1303G>A and c.1586G>A in cDNA clones and found them to be recurrent in a series of 21 umbilical cord blood mononuclear cell (CBMC) samples studied. Two less conserved U-to-C changes were also observed. These alternative changes were found to be significantly higher in non-progenitor as compared to progenitor CBMCs, while they were found to be absent in a series of AML samples studied, indicating they are targeted, cell type-specific mRNA editing modifications. Since APOBEC/ADAR family members are implicated in RNA/DNA editing, we screened them by RNA-interference (RNAi) for WT1-mRNA changes and observed near complete reversal of WT1 c.1303G>A alteration upon APOBEC3A (A3A) knockdown. The role of A3A in mediating this change was confirmed by A3A overexpression in Fujioka cells, which led to a significant increase in WT1 c.1303G>A mRNA editing. Non-progenitor CBMCs showed correspondingly higher levels of A3A-mRNA and protein as compared to the progenitor ones. To our knowledge, this is the first report of mRNA modifying activity for an APOBEC3 protein and implicates A3A in a novel G-to-A form of editing. These findings open the way to further investigations into the mechanisms of other potential mRNA changes, which will help to redefine the RNA editing paradigm in both health and disease.Classic deamination mRNA changes, including cytidine to uridine (C-to-U) and adenosine to inosine (A-to-I), are important exceptions to the central dogma and lead to significant alterations in gene transcripts and products. Although there are a few reports of non-classic mRNA alterations, as yet there is no molecular explanation for these alternative changes.  APOB (apolipoprotein B) transcripts, changing the Gln codon (CAA) to a nonsense codon (UAA), and leading to ApoB48 rather than ApoB100 variant[in vitro polymerase reactions. While APOBEC1 knockout leads to defects which are not incompatible with development[According to the central dogma, genetic information is faithfully transferred from DNA to mRNA to protein. Classic mRNA editing in both plant and animal cells is an important exception where mRNA is altered during or after transcription. In human cells, APOBEC1  was first recognised to alter cytidine to uridine (C-to-U) in 0 variant. Since ivelopment, ADAR1 ivelopment. Other ABoth classic editing events in mammals comprising A-to-I and C-to-U alterations are mediated by nucleoside deamination reactions. Although there are some reports of reverse U-to-C, 7 as weWT1 transcripts[WT1 mutations affecting the zinc finger (ZnF) domains are implicated in Wilms tumor[WT1 variants in AML and CBMCs, we observed recurrent G-to-A and occasional T-to-C changes in WT1-cDNA from CBMCs. We first demonstrated that such alternative changes were true cell type-specific mRNA modifications, rather than in vitro phenomena. Next, we hypothesized that known RNA/DNA editors might be implicated in these modifications. Hence, we used these novel changes in WT1 as a marker and assessed how they were affected by APOBEC/ADAR knockdown. The results were confirmed by overexpression studies.Non-classic U-to-C mRNA editing was first reported in anscripts. WT1 is anscripts). In addlms tumor and acutlms tumor. While sg for 30 minutes at 20°C. The middle buffy coat layer containing the CBMCs was transferred to a new tube and washed with PBS containing 2% fetal bovine serum (FBS), before spinning at 300 X g for 7 minutes at 4°C. Red cell lysis was performed using cold ammonium chloride. Viable cells were counted by Trypan blue exclusion on a Neubauer hemocytometer.Human umbilical cord blood samples were collected from normal deliveries in the Royal London Hospital after obtaining written and signed informed consent, and with the project approved by the East London Research Ethics Committee. The samples were processed separately (Cb samples), or up to five samples were pooled for some experiments (Cbp samples). Blood was diluted using 2 volumes of phosphate buffered saline (PBS) before being layered on half volume of Ficoll in 50 ml tubes and centrifuged at 360 X Progenitor (lineage-marker negative) CBMCs were separated from non-progenitor (lineage-marker positive) cells using the StemSep Human Progenitor Enrichment Kit (StemCell Technologies) according to the manufacturer’s protocol. Briefly, CBMCs were incubated with a cocktail of antibodies against human hematopoietic lineage markers, followed by incubation with a magnetic colloid. The cell suspension was then pumped through a negative selection column mounted in a magnetic stand. The progenitor CBMCs were collected at the tubing outlet, and the non-progenitor CBMCs were separately washed through the column after removal from the magnetic stand. Cells were cryopreserved in 10% DMSO / 90% FBS at -80°C for later use.Peripheral blood or bone marrow samples were collected from AML patients attending St Bartholomew’s Hospital, London, after obtaining written and signed informed consent. Established myeloid cell lines THP1, Fujioka6 cells using DNeasy Blood & Tissue Kit (Qiagen) according to manufacturer’s protocol. Total RNA was also extracted from up to 5 x 105 and 5–50 x 105 cells using RNeasy Micro and Mini Kits (Qiagen), respectively, according to manufacturer’s protocols. All RNA samples were treated with DNase, as recommended by the manufacturer.Genomic DNA was extracted from 10WT1 exon 7 was amplified using 20–50 ng of gDNA, 200 nM Ws-X7f5 , making it ready for cloning or direct Sanger sequencing.20, and 500 μM dNTP mix. WT1-cDNA was amplified using a mixture of 2 μl cDNA, 300 nM Ws-Ex6f (see cDNA was made according to the SuperScript III First-Strand Synthesis protocol (Life Technologies) using up to 5 μg total RNA, 2.5 μM oligo(dT)A3A and GAPDH  mRNA levels were assessed using Taqman Gene Expression assays  according to manufacturer’s protocol. A3A and GAPDH mRNA levels were assessed in triplicate. The Life Technologies 7900HT Fast Real-Time PCR System was used for amplification, and the results were analyzed using SDS software .E. coli was spread on LB agar plates at 37°C overnight. Between 30 to 50 colonies per RT-PCR product or 280 colonies per PCR product were randomly picked and inoculated into 1 ml LB media in 96-well blocks, and incubated in a shaker at 37°C overnight for plasmid preparation. In order to assess other hematopoietic transcripts in CBMCs and also WT1-cDNA in AML cells, at least 10 colonies were examined per sample. The plasmids were extracted using Nucleospin Robot-96 Plasmid Core Kit (ClonTech) on a BioRobot 9600 machine (Qiagen).Gel purified PCR or RT-PCR products were cloned using TOPO XL PCR Cloning Kit (Life Technologies) according to manufacturer’s instructions. Transformed Direct Sanger sequencing was performed using gel-purified PCR/ RT-PCR products, the same amplification primers, and BigDye Terminator kit , for 25 cycles. The amplified gDNA/ cDNA was analyzed using a 3730xl BioAnalyzer (Life Technologies) after dye-exclusion with Chemagic SEQ Pure Kit on a Biomek FX machine (Beckman Coulter). Sanger sequencing of the gDNA/ cDNA clones was performed using the same protocol, but with miniprep plasmids and universal M13f/ M13r primers for the cycling reaction.www.dharmacon.com/designcenter) and two additional siRNAs for A3A. The siRNAs were selected using the following criteria: a GC ratio of 30–64%; preferential targeting of the open reading frame (ORF); affecting as many variants as possible; highest predicted efficiency; at least two mismatches for each of the antisense and sense strands; and low frequency of seed region (sense nucleotides 2 to 8) matching to 3’UTRs throughout the genome  was designed for each of 14 candidate genes using Dharmacon’s siDesign Center  and incubated at 37°C and 5% CO2 overnight. For silencing experiments, a mixture of 3 μl test or control siRNA 20 μM , 3 μl Alx-si 20 μM (Sigma-Aldrich), and 5 μl HiPerfect Transfection Reagent (Qiagen) was added to 8 x 105 CBMCs in 109 μl 5% FBS-IMDM in a 24-well plate and incubated at 37°C and 5% CO2 for 6 hours. Then, they were topped up using 480 μl pre-warmed 5% FBS-IMDM and incubated as above. The plates were examined using fluorescent microscopy for transfection efficiency after 24 hours. Efficiently transfected samples were harvested 48 hours after the transfection, and their total RNAs extracted immediately as described above.Pooled or separate CBMC samples were resuspended at a density of 5 x 10A3A overexpression (RG220995) and empty pCMV6-AC-GFP (PS100010) vectors were obtained from OriGene. Overexpression was performed using the Amaxa Cell Line Nucleofector Kit V (Lonza) with 3.5ug DNA per 2 million cells. GFP positive cells were sorted on a Cytomation MoFlo or a BD FACS Aria sorter.Green-fluorescent protein (GFP)-tagged Protein was extracted using RIPA buffer containing protease inhibitor and DTT (Sigma-Aldrich). Western blots were performed using a Novex Mini Cell gel tank and Novex gels (Life Technologies), and semi-dry transfers using Hybond ECL membrane . Protein was detected using rabbit anti-APOBEC3A (Santa Cruz Biotechnology) and mouse anti-beta-Actin (Sigma-Aldrich), followed by HRP-conjugated goat anti-rabbit and anti-mouse secondary antibodies (Sigma-Aldrich), respectively. Detection was performed using ECL Plus . Relative protein quantification was performed using Image J.A% value at c.1303 was obtained by counting the A/G in sequenced clones and performing the calculation 100 x A / (A + G). Fisher’s Exact test  was used to compare the A% at c.1303 between each experiment and the control, and also for alterations in progenitor/ non-progenitor subpopulations. For association analyses, under a hypothesis of independence of changes occurring at the two sites, the count data for changes at neither, only at site 1, only at site 2, and at both sites follow a multinomial distribution. We estimated the independent probabilities from the observed data  and provided the R function dmultinom with these estimates to assess the probability of the observed data under this null.WT1-cDNA . This excluded the possibility of the classic C-to-U and A-to-G alterations in an antisense strand as a cause of observed G-to-A and U-to-C changes, respectively. We tested the possibility of minor primary genomic changes samples with prominent c.1586G>A alteration, which did not show any genomic DNA (gDNA) changes in 280 copies. In order to validate the alternative modifications in a larger series of CBMCs, we later examined the same WT1 region using Sanger sequencing of paired gDNA-cDNA from a series of 21 CBMC samples  and alternative (1586G>A) changes to be significantly associated with canonical KTS splicing variants (cis) with c.1528A>G ones  was also seen at c.1644 in two control samples. Moreover, some non-conserved G-to-A alterations were also seen in knockdown experiments, including those at c.1581 using A3G-si (13%) and at c.1586 using A3C-si (88%)  .. Hence,si (88%) , with noA3A-specific, we knocked down the gene using two additional specific siRNAs, alongside A3A-si1. Assessment of A3A mRNA level in the A3A-silenced CBMC samples showed 86% to 98% silencing efficiency for the tested A3A-siRNAs  at c.1303  transcripts in both malignant and normal colorectal samples[TPH2 (tryptophan hydroxylase 2) transcripts[Ganoderma lucidum by RNA-sequencing[Non-classic G-to-A mRNA changes were first reported in l samples, but latanscripts. G-to-A equencing. Transamequencing. Howeverequencing, and manequencing, includiequencing and systequencing, 23.WT1 transcripts at c.1303 and c.1586, which are of potential functional significance. The former corresponds to polymorphic site rs16754, which is a located in a hotspot for most WT1 mutations in AML[WT1 expression[in vitro phenomenon, there is yet no known enzyme performing these novel changes.Herein we report novel G-to-A mRNA changes in xpression. Moreovexpression and poorCurrently, APOBEC1 (A1), which classically deaminates cytidine to uridine (C-to-U), is the only known mRNA editor among APOBEC/AID family. Other fWT1-mRNA alterations revealed a pattern of associated alternative-classic events, including mutual cis G-to-A and C-to-U changes (P = 2.6 x 10-7). Hence, we hypothesized that this cis position might suggest a role for classic mRNA/DNA editors in alternative editing. RNAi screening of the APOBEC/ADAR genes suggested A3A as the potentially implicated gene, and repeat experiments using two additional A3A-siRNAs led to similar results. These results were further tested by A3A overexpression in Fujioka cells, with relatively low A3A expression and informative AG genotype at c.1303, which led to a significant 1.5 fold increase in A% at that site. These findings confirm that the G-to-A class of alternative mRNA editing is specifically mediated by an A3A-dependent mechanism. However, the modest increase in G-to-A editing after A3A overexpression despite its almost complete reversal in RNAi studies may suggest a role for potential cofactors, as required by deaminases[Our clonal analysis of  changes . as compared to progenitors. This was in line with differential G-to-A editing activity observed in mentioned subpopulations  family, consisting of seven proteins with one or two cytidinedeaminase (CDA) domains, have be1 and Alu, 42, and1 and Alu. Intrigu1 and Alu, 44, a p1 and Alu. Finallyrmutation and the rmutation.WT1 mRNA which correspond to driver cancer mutations suggests a potential role for this novel modification in cancer mutagenesis. In fact, in a recent global study assessing 14 different cancer types, A3B and A3A expression was found to be significantly higher in those samples with an APOBEC mutation type, particularly in breast cancer samples[A3B and A3A expression levels correlated to the number of APOBEC mutations among different cancers studied[This novel function of A3A as a dual DNA-mRNA modifier raises broader questions of the role of mRNA editing, and specifically the role of A3A, in cancer. The fact that A3A induces particular changes in r samples. Follow-s studied. Functios studied. These sWT1 variants. More importantly, the G-to-A editing was specifically mediated by A3A, which to our knowledge, is the first report of a mechanism for alternative mRNA editing and furthermore of a function that has not previously been described for any of the APOBEC3 proteins. The implication of A3A in both mRNA and DNA alterations and its apparent up-regulation in different cancers raises the possibility that aberrations in alternative mRNA editing are involved in cancers. This is not only an intriguing question, but also a potential target for therapy. Moreover, our work demonstrates an approach to identify genes which mediate other potential novel RNA changes detected by modern high-throughput approaches. Finally, this study provides evidence against exclusion of the potential RNA editing sites which are simply coinciding with polymorphic sites in high-throughput sequencing studies.In summary, this study confirms the existence of two non-classic forms of mRNA editing in human, comprising G-to-A and U-to-C changes, using targeted Sanger sequencing rather than next-generation sequencing with its inherent potential for errors due to misalignment. These changes are likely to be of functional consequence in view of their cell specific nature, and in the case of G-to-A changes, their association with canonical S1 FigWT1-cDNA clones, and the chromatograms were aligned and examined for potential changes at c.1586, which did not show any alterations.Nineteen AML samples were examined using Sanger sequencing of the (TIF)Click here for additional data file.S2 FigKnockdown experiments of the candidate genes were associated with some G-to-A alterations at non-conserved sites which were not observed in the control experiments, including changes at c.1581 (A) and c.1586 (B) associated with A3G-si and A3C-si, respectively.(TIF)Click here for additional data file.S3 FigWT1 transcripts. B. Expression levels of A3A in corresponding myeloid cell lines compared to b-Actin as determined by Western blotting.A. A% of at c.1303 in four myeloid cell lines as determined by clonal sequencing of the (TIF)Click here for additional data file.S4 FigWT1-cDNA clones are aligned and examined for the changes occurring in the same clones, i.e. in cis, with one altered chromatogram expanded for each of the associated events. Statistically significant associations are found for c.1388U>C and c.1528A>G (A), as well as c.1505C>U and c.1609G>A (B and C). *** P < 0.001.Sanger sequencing chromatograms of the (TIF)Click here for additional data file.S5 FigClassic mRNA editing is explained by deamination reactions converting C to U (A) and A to I, which mimics G (B), while proposed alternative mRNA editing model involves amination reactions converting U to C (C) and G to D, mimicking A (D). Each edited base is shown with its paired base.(TIF)Click here for additional data file.S1 TableGross G-to-A changes were seen in seven informative samples at c.1303 (underlined), including three homozygous GG samples  with prominent A at c.1303, not explained by imbalanced allele expression. Sixteen samples showed prominent A at c.1586 (underlined). Overall, 17 samples showed G-to-A change at c.1303 and/or c.1586. * Samples with additional T-to-C alterations at c.1388 and c.1402.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.