MYB, MYC and CCND1. As a consequence, knockdown of JMJD2B severely impairs estrogen-induced cell proliferation and the tumor formation capacity of breast cancer cells. Furthermore, Jmjd2b-deletion in mammary epithelial cells exhibits delayed mammary gland development in female mice. Taken together, these findings suggest an essential role for JMJD2B in the estrogen signaling, and identify JMJD2B as a potential therapeutic target in breast cancer.Estrogen is a key regulator of normal function of female reproductive system and plays a pivotal role in the development and progression of breast cancer. Here, we demonstrate that JMJD2B  constitutes a key component of the estrogen signaling pathway. JMJD2B is expressed in a high proportion of human breast tumors, and that expression levels significantly correlate with estrogen receptor (ER) positivity. In addition, 17-beta-estradiol (E2) induces JMJD2B expression in an ERα dependent manner. JMJD2B interacts with ERα and components of the SWI/SNF-B chromatin remodeling complex. JMJD2B is recruited to ERα target sites, demethylates H3K9me3 and facilitates transcription of ER responsive genes including  Estrogen plays an important role in normal physiology and several human diseases including breast cancer. The estrogen signaling pathway is a reliable therapeutic target for estrogen-receptor (ER) positive subtype of breast cancer. Understanding of how ER regulates transcription is key to overcoming resistance to existing selective ER modulators (SERMs) and identifying non-SERM targets suitable for novel therapeutic approaches The biological functions of estrogen are mediated through ER, which regulates transcription of ER target genes by binding to estrogen responsive elements (EREs) Genome-wide analyses of chromatin modifications have revealed a complex landscape of modified histones at transcription start sites (TSSs), distal regulatory elements and conserved sequences. In general, methylated H3K4 and H3K36 are associated with active transcription, whereas methylated H3K9, H3K27 and H4K20 are associated with gene silencing. H3K4me3 and H3K27me3 signals peak near TSSs. The relative presence of H3K4me3 and H3K27me3 has been shown to affect promoter activity in vivo is still unclear.Recent discoveries of histone demethylases have advanced our understanding of transcriptional regulation Abnormalities in the methylation of histones by histone methyltransferases have been implicated in various cancers All experimental procedures were approved by the local Ethical Committee for Animal Experimentation of Ontario Cancer Institute (OCI), University Health Network (AUP#2031). The research projects that are approved by the local Ethical Committee for Animal Experimentation of OCI are operated in accordance with applicable Federal, Provincial, Municipal and Institutional regulations, the Policies and Guidelines of the Canadian Council on Animal Care and the Province of Ontario's Animals for Research Act. The animals are housed – accordance with applicable Federal, Provincial, Municipal and Institutional regulations, the Policies and Guidelines of the Canadian Council on Animal Care and the Province of Ontario's Animals for Research Act in the OCI Animal Facilities of the university. The Institute is committed to the highest ethical standards of care for animals used for the purpose of continued progress in the field of human medicine.All antibodies and primers used are described in http://www.oncomine.org) JMJD2B mRNA expression in human breast cancers. Statistical analysis of the differences in JMJD2B expression between ER-positive and ER-negative breast cancers was performed using ONCOMINE algorithms that allow for multiple comparisons among different studies.The ONCOMINE database and gene microarray analysis tool, a repository for published complementary DNA microarray data . MCF-7 cells were cultured in Eagle's Minimum Essential Medium (ATCC #30-2003) supplemented with antibiotics, 10% fetal bovine serum (FBS) and 0.01 mg/mL bovine insulin. T-47D cells were cultured in RPMI-1640 supplemented with antibiotics, 10% FBS and 0.2 units/mL bovine insulin. For steroid-free medium, phenol red-free DMEM (Gibco) and charcoal/dextran-treated FBS  were used.RNA was extracted from cells using an RNeasy Mini kit (Qiagen). Total cellular RNA was converted into cDNA by reverse transcription  using random primers. PCR amplification was performed using Power SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen Life Technologies) through 40 cycles of 95°C for 15 s and 60°C for 60 s using an Applied Biosystems PRISM 7900 Sequence Detection System.siRNAs for JMJD2A and JMJD2B and non-targeted control siRNA were purchased from Dharmacon. Validations of the siRNAs are in 5 cells in culture medium plus 0.35% agar and 1 ng/mL puromycin. Medium alone was added as the top layer to prevent desiccation of the agarose. Colonies were allowed to form for 14 days prior to visualization by crystal violet staining.ZR-75-1 cells, MCF-7 cells, or derivative cell lines were cultured in 12-well plates containing a bottom agar layer consisting of culture medium plus 0.7% agar and 2 ng/mL puromycin. The middle layer contained 10bgFoxn1nuBtkxid, 7-week old, Charles River Laboratories) three days before tumor transplantation. 3×106 ZR-75-1 cells grown in cell culture were suspended in 50 µL medium, mixed with 50 µL Matrigel, and injected subcutaneously into hind flanks. Tumors derived from injected cells were harvested two weeks after transplantation.Slow-release estradiol pellets were implanted subcutaneously into NIH-III mice , cells were pulsed with 10 µM BrdU for 1 hr. The FITC BrdU flow kit (BD Biosciences) was used to detect BrdU. Fluoresence-activated cell sorting (FACS) analysis was performed on a FACSCalibur (Becton Dickinson), and data were analyzed with Cellquest or FlowJoe software.2, 0.5% NP-40) in the presence of protease inhibitors (Complete(R), Roche). After washing with PBS, the pellet was resuspended in 1 mL of NP-40 IP lysis buffer (50 mM TrisHCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1 mM EDTA) containing protease inhibitors. The lysate was sonicated at 5% output for 20 sec. After centrifugation, the supernatant was collected and precleared by incubating with protein A magnetic beads  at 4°C for 1 hr. Fresh protein A beads were washed with PBS-T (PBS+0.05% Tween) three times, resuspended in 100 µL PBS-T, and incubated with 5 µg of indicated antibodies at 4°C for 1 hr. The antibody-coated beads were then washed with PBS-T three times and incubated with 800 µL of the lysate at 4°C over night. The rest of the samples were kept for INPUT. After extensive wash, the beads were suspended in 80 µL of 2× sample buffer and boiled at 95°C for 5 min to elute associated proteins. To elute JMJD2C, the beads were incubated with 40 µL of 8 M Urea for 15 min at 37°C, and then boiled with 40 µL of 4× sample buffer.Cells were washed with PBS, scraped off, and collected by centrifugation. The cells were suspended in hypotonic buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgClCells were crosslinked in 1% formaldehyde for 10 min at room temperature. Cells were lysed and sonicated to obtain fragmented chromatin samples, which were immunoprecipitated using specific antibodies coupled to protein A Dynabeads (Invitrogen). The protein-DNA complexes were eluted from the beads and incubated at 65°C overnight to reverse protein-DNA crosslinks. Precipitated DNA was analyzed by real-time PCR. Refer to http://research4.dfci.harvard.edu/brownlab/datasets;  http://www.ncbi.nlm.nih.gov/pubmed/19005469) using UCSC genome browser. ERα responsive elements (EREs) were determined by Mapper (http://bio.chip.org/mapper).Putative ER binding sites were identified by analyzing the bed files  to obtain Lin− cells.Mammary fat pads were minced into paste, digested in collagenase/hyaluronidase solution , and dissociated with dispase . Samples were washed with Hanks' Balanced Salt Solution supplemented with 2% FBS and 2 mmol/L EDTA (HFE) and centrifuged at 300×JMJD2B mRNA was higher in ER-positive cancers than in ER-negative cancers  to target genes upon estrogen stimulation. We found that JMJD2A, JMJD2B, and JMJD2C interacted with one of the core subunits of SWI/SNF complex, BRG1 ATPase . JMJD2A GREB1 in response to E2 was significantly reduced in JMJD2B-depleted T-47D cells and JMJD2B-depleted MCF-7 cells but not in JMJD2A-depleted cells ), control MCF-7 cells treated with E2 (control E2(+)), steroid-depleted JMJD2B-depleted MCF-7 cells (siJ2B E2(−)), and JMJD2B-depleted MCF-7 cells treated with E2 (siJ2B E2(+)). After normalizing the data, we identified genes that exhibited a log-fold change of greater than 2 between control E2(−) and control E2(+) cells, or between control E2(+) and siJ2B E2(+) cells.  was set at <0.05.) In total, we identified 1432 differentially expressed genes , S3, S4.ed cells , althougMYB, MYC, and CCND1 genes were reduced in JMJD2B-depleted cells upon E2 treatment, impairment of G1/S transition appears to contribute the defective proliferation. It has been recently reported that JMJD2B-depletion also influences genes required for G2/M transition To determine whether JMJD2B is required for cellular responses to estrogen, we stimulated JMJD2B-depleted T-47D and MCF-7 cells with E2 and measured BrdU incorporation at 72 hr post-transfection. E2 stimulated the proliferation of control cells but had a limited effect on the proliferation of JMJD2B-depleted cells  and S4D,MYB, JMJD2B, and GREB1 . We selected candidate ER binding sites according to the results of a published genome-wide analysis nd GREB1 . Recentlive TSSs . The intive TSSs . We alsoive TSSs . NotablyMYB ERE appears to decrease over the time after E2 addition but H3 displacement during the time window we have analyzed is not statistically significant whereas H3K9me3/H3 ratio at MYB ERE decreases to approximately one-third in the presence of E2  by expression of Cre recombinase under the control of the MMTV promoter  MECs lox mice . These dJmjd2b-deficiency on proliferation of MECs, the isolated Jmjd2bflox/flox MECs were infected with either Mock-GFP (control) or iCre-IRES-GFP retrovirus, and then cultured in vitro. Western blotting confirmed that Jmjd2b protein was absent from cells infected with Cre-expressing virus , indicating that Jmjd2b is not a general regulator of cell proliferation. These observations further support our findings that Jmjd2b has tissue and signal specific functions.It is noteworthy that we have obtained viable Intensive studies of how nuclear receptors transmit ligand binding signals have identified several distinct multiprotein complexes. These complexes are recruited to promoters and enhancers, and modulate higher-order chromatin and nucleosomal structures leading to activation or repression of target genes. Genome-wide analysis of ERα binding sites has been performed by numerous groups and potential ERE sites have been mapped in promoter, enhancer and intronic regions; however, how the distal regulatory EREs function and what are co-factors at these sites remain to be elucidated.in vivo  T-47D cells were transfected with siRNA against JMJD2A (target sequences #1 and #2). (B) T-47D cells were transfected with siRNA against JMJD2B . (A and B) Total cell lysate was prepared 72 hr after transfection and subjected to western blotting. (C) T-47D cells were transfected with si JMJD2B #1 or #2. RNA was extracted 72 hr after transfection and subjected to quantitation by real-time RT-PCR. (D) MCF-7 cells were infected with a lentiviral vector harboring JMJD2B shRNA targeting sequence #1, #2 or #3. Total cell lysates were prepared 48 hr after infection and subjected to western blotting. (E) T-47D cells were transfected with siRNA against JMJD2B . Total cell lysate was prepared 72 hr after transfection and subjected to western blotting for JMJD2A. The introduction of #6 siRNA reduced JMJD2A protein levels. We therefore used #1 and #2 siRNA in this study. Positive control: protein samples from JMJD2A overexpressed cells. (F) T-47D cells were transfected with siRNA against JMJD2A . Total cell lysate was prepared 72 hr after transfection and subjected to western blotting for JMJD2B.(TIF)Click here for additional data file.Figure S2JMJD2B knockdown decreases colony formation of MCF-7 cells. Single cell suspensions of MCF-7 cells expressing control shRNA or shRNA against JMJD2B (target sequence #2) were seeded in 0.35% soft agar. After 14 days, colonies were stained with crystal violet. Microscopic fields were photographed (left). The number of colonies/cm2 was determined (right). Data are the mean number of colonies formed in three wells ± s.d., p<0.01. A representative result from four independent trials.(TIF)Click here for additional data file.Figure S3JMJD2B interacts with ERα and SWI/SNF-B complex. (A) Catalytic activity of JMJD2B does not influence the interaction with ER. 293T cells were co-transfected with MYC-ERα and either empty vector, FLAG-JMJD2B or FLAG-ΔFeJMJD2B (a mutant with (H189Y and E191A) point mutations in the iron-binding region). α-MYC immunoprecipitates were analyzed by western blot with α-FLAG antibody. (B) Benzonase treatment does not affect the interaction of endogenous JMJD2B and ER. Nuclear lysates of T-47D cells were lyzed and sonicated in the presence or absence of 500 unit of Benzonase (Novagen) before immunoprecipitation with α-JMJD2B Ab. Input lysate and immunoprecipitated samples were immunoblotted using α-ERα antibody. Full-length blots are presented in (TIF)Click here for additional data file.Figure S4JMJD2B mediates induction of ER target genes and estrogen-dependent proliferation of breast cancer cells. (A) JMJD2B is required for the induction of ER target genes. MCF-7 cells were transfected with either control siRNA or JMJD2B siRNA (target sequence #1), cultured in steroid-free medium for 72 hr, and stimulated with or without E2 for 4 hr. mRNA levels of JMJD2B or the indicated ER target genes were measured by real-time RT-PCR. Results shown are mean mRNA level normalized to the amount of ACTB mRNA ± s.d. of triplicates. **, p<0.01. (B) Microarray data for representative ER target genes. Signal intensities on microarray for representative ER target genes are shown by their mean values (±SD). (C) Heat map representation of differentially expressed genes. One thousand four hundred and thirty-two differentially expressed genes  were sorted by hierarchical clustering. Each row represents a gene and each column represents a sample. Red indicates higher expression and blue lower expression. (D) JMJD2B knockdown impairs cellular response to estrogen. T-47D cells or MCF-7 cells transfected with control siRNA or JMJD2B siRNA were cultured in steroid-free medium for 48 hr, stimulated for 24 hr with E2, labeled for 1 hr with BrdU, and stained with anti-BrdU antibody and 7-AAD. The fraction of BrdU-positive cells was determined by flow cytometry. A representative result from three independent experiments.(TIF)Click here for additional data file.Figure S5ER binding sites assessed in this study. (A) The UCSC Genome browse map depicts the ER binding sites of MYB locus, JMJD2B locus, and GREB1 locus. Depicted ER binding sites are identified in previously published studies. (B) Effect of JMJD2B knockdown on induction of ER target genes. mRNA levels of JMJD2B or the indicated ER target genes in the corresponding samples applied for the ChIP analysis are shown. (C) The change of H3 and H3K9me3/H3 ratio at Myb ERE.(TIF)Click here for additional data file.Figure S6Conditional gene targeting of murine Jmjd2b locus. (A) Introduction of loxP sites into Jmjd2b. A portion of the murine wild type Jmjd2b locus showing exon 5 (grey rectangle) and an 11.9 kb XbaI fragment are shown at the top. The targeting vector was designed to generate floxed exon5 , to flank the PGK-neo cassette with frt sequences (grey triangles), and to introduce a new XbaI site (X). The targeted allele contains a diagnostic 7.7 kb XbaI fragment. The neo cassette was removed in vivo by FLPe recombinase as described in Jmjd2b exon 5. The position of the 5′ flanking probe used for genotyping is indicated. (B) Southern blot analyses to identify Jmjd2bneo/+ ES cells . Genomic DNA was digested with XbaI (upper panel) or EcoRI (lower panel) and hybridized with the probe indicated in (A). (C) Southern blot analyses to identify Jmjd2bneo/+ mice (lanes 2 and 3), Jmjd2bflox/+ mice (lanes 4 and 5), and Jmjd2b+/Δ mice (lanes 6 and 7). Genomic DNA was digested with XbaI (upper panel) or EcoRI (lower panel) and hybridized with the probe indicated in (A). The numbers in brackets indicate the ES clones from which the mice were derived.(TIF)Click here for additional data file.Figure S7Full-length blots images. Full-length blot images related to (TIF)Click here for additional data file.Table S1P value of difference in JMJD2B mRNA expression in ER-positive and ER-negative breast cancers in 19 ONCOMINE studies.(DOC)Click here for additional data file.Table S2Genes down regulated in JMJD2B depleted cells.(XLS)Click here for additional data file.Table S3Genes upregulated in JMJD2B depleted cells.(XLS)Click here for additional data file.Table S4Genes down regulated in E2 treated JMJD2B depleted cells.(XLS)Click here for additional data file.Table S5Genes upregulated in E2 treated JMJD2B depleted cells.(XLS)Click here for additional data file.Procedure S1(DOC)Click here for additional data file.


To test this hypothesis we applied a unilateral ureteral obstruction (UUO) model of RIF on A2AR knockout mice and their wild-type littermates, combined with the intervention of a selective A2AR agonist, CGS 21680. On days 3, 7 and 14 post-UUO we evaluated the effects of A2AR manipulation on the molecular pathological progresses of RIF, including the cellular component of interstitial infiltration, expression of profibrotic factors, cellular biomarkers of EMT, and collagen deposition of extracellular matrix. Our data demonstrated that activation of A2AR significantly suppressed the deposition of collagen types I and III, reduced the infiltration of CD4+ T lymphocytes, and attenuated the expression of TGF-β1 and ROCK1, which in turn inhibited and postponed the EMT progress. Conversely, genetic inactivation of A2AR exacerbated the aforementioned pathological processes of UUO-induced RIF. Together, activation of A2AR effectively alleviated EMT and RIF in mice, suggesting A2AR as a potential therapeutic target for the treatment of RIF.Renal interstitial fibrosis (RIF) is the common pathological process of chronic kidney diseases leading inevitably to renal function deterioration. RIF and its preceding epithelial-mesenchymal transition (EMT) are commonly triggered by an early occurring renal inflammation. However, an effective approach to prevent EMT and RIF is still lacking and of urgent need. Recently, the adenosine A Regardless of the etiology, almost all forms of end-stage renal disease share the common pathological feature of progressive renal interstitial fibrosis (RIF) and tubular atrophy 2A receptor (A2AR) emerges as a novel inflammation regulator affecting the inflammation process and tissue repair. Pharmacology studies showed that A2AR agonist, CGS21680 and ATL193, can effectively suppress inflammation 2AR leads to attenuation of glomerulonephritis and renal injury 2AR activation inhibits Rho/ROCK1 in hepatic stellate cells 2AR manipulation plays an important regulatory role in inflammation and may also affect EMT event. Therefore, we hypothesize that activation of A2AR may suppress cellular infiltration, EMT event and profibrogenic factors, thereby preventing consequent pathology of RIF. Conversely, inactivation of A2AR may lead exacerbation of RIF.Recently, adenosine AA unilateral ureteral obstruction (UUO) model has been refined to elucidate the pathogenesis and mechanisms responsible for RIF 2AR-based manipulation on several aspects of RIF progression, including interstitial lymphocyte infiltration, cellular biomarkers of EMT, expression of the profibrogenic factor TGF-β1 and its downstream Rho/ROCK1 pathway, as well as the consequent extracellular matrix accumulation.Using the experimental UUO-induced RIF mouse model, the present study was aimed to evaluate the modulatory effect of A2AR knockout (KO) mice (A2AR−/−) and their wild-type (WT) controls (A2AR+/+) were bred at the Animal Care Center of the Research Institute of Surgery of TMMU after being imported from Boston University School of Medicine as previously described 2AR KO mice and their WT littermates were randomly designated into six experimental groups  and performed under the supervision of the facility veterinary staff. Mice used in the present study, i.e., genetic Aoups see  accordinMice (20–25 g weight) were subjected to the UUO procedure under anesthesia as previously described 2AR was induced by daily systemic administration of the selective A2AR agonist, CGS 21680  from day 1 after UUO through the designed experimental time-points, i.e., day 3, 7, and 14 after UUO, when mice were scarified and their kidneys were harvested.Pharmacological activation of A2AR, TGF-β1, and ROCK1 mRNAs using SYBR Premix Ex Taq kit  and a qPCR reaction thermal cycle of 40 cycles of 95°C (30 s), 58°C (30 s), and 70°C (30 s). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control to normalize RT-qPCR readout. Gene mRNA expression levels were calculated relative to the expression level of GAPDH. All primers used  and the reverse transcription reaction was performed using SYBR Premix Ex Taq kit , according to the manufacturer's instructions. Then qPCR was performed to quantify the expression level of A2AR , anti-CD3 , anti-CD4 , anti-CD8 , anti-Foxp3 , anti-CD11b , anti-CD68 , anti-F4/80 , anti-collagen I , anti-collagen III , anti-α-SMA . All the quantitative morphological analyses were performed by separate investigator who was blinded to the treatment of samples. Positive stained cells and/or area were assessed and expressed as integrated optical density (IOD) or area. Three sections of each mouse kidney were measured, and 10 random fields or appointed area around vessels were chosen and calculated under magnification of 100x (for CD4) or 400x . The IOD or positive area was acquired by the Image-Pro Plus 6.0 program .Hematoxylin and eosin (H&E) staining and all immunohistostainings were performed on 4 μm-thick paraffin-embedded slice of kidney using a similar procedure as previously described The Western blot was performed according as previously described post hoc comparisons. P<0.05 was considered statistical significance.The data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni 2AR on renal fibrosis, we applied the UUO model to mice combined with A2AR agonist CGS21680 and genetic A2AR inactivation (as aforementioned paradigm in Methods). Pathology assessment using H&E staining and immunohistostaining of Col I and Col III deposition were evaluated at day 3, 7, and 14 after UUO. Our H&E data demonstrated the successfulness of UUO modeling with featured pathological changes, e.g. progressively aggravated tubular dilatation and leukocytes infiltration , but devoid in KO mice (KO+Sham)  mice remained under a detectable threshold from day 1 throughout day 14 post-UUO  . Furtherpost-UUO .2AR agonist-treated WT+UUO+CGS group , compared to WT+UUO+Veh group  mice, showing an increased Col I and Col III levels  in mouse kidneys from WT+UUO+Veh group  and 3, i2AR agonist CGS21680 treatment (in WT+UUO+CGS group) reversed deposition of collagens at day 3 and day 7 post-UUO, compared to WT+UUO+Veh group . Nevertheless, activation of A2AR effectively attenuated and postponed the progression of RIF whereas inactivation of A2AR exacerbated the RIF process.Importantly, A2AR-mediated effects on the EMT process we detected the expression levels of α-SMA (the myofibroblast marker) and E-cadherin  that indicate the transdifferentiation status of epithelial to myofibroblast. Western blot assay showed that at day 3 no expression difference of α-SMA and E-cadherin was found between each of the UUO groups and sham groups  compared to WT+UUO+Veh group, P<0.05, n = 5 per group, 2AR agonist treatment enhanced E-cadherin level in WT+UUO+CGS group (by 27.9% at day 7 and by 20.6% at day 14) compared to WT+UUO+Veh group  led to an opposite effect on α-SMA and E-cadherin levels compared to A2AR activation (WT+UUO+CGS) treatment. The expression of α-SMA was enhanced by 17.9% (day 7) and 54.2% (day 14), whereas the E-cadherin levels were decreased by 15.7% (day 7) and 39.6% (day 14), compared with WT+UUO+Veh group, , but devoid in WT mice which received CGS21680 treatment (WT+UUO+CGS), at day 7 post-UUO  and Apost-UUO . This im2AR modulation on RIF, we detected the mRNA expression of two crucial profibrotic mediators, TGF-β1 and ROCK1 using RT-qPCR. We showed that the expression level of TGF-β1 mRNA was significantly increased at day 3 through day 14 in WT+UUO+Veh group  compared to WT+Sham control group  and 30.0% (P<0.05) at day 3 and day 7, respectively, vs. WT+UUO+Veh group (n =  10 per group). Conversely, genetic inactivation of A2AR in KO+UUO+Veh group led to an additional enhancement in mRNA expression of TGF-β1, by 39.1% (day 3) and 37.5% (day 7) compared to WT+UUO+Veh group, correspondingly  led to an exacerbated enhancement of ROCK1 level, by 30.0% (day 3) and 17.1% (day 7) vs. WT+UUO+Veh , showing an increase of 37.8% (day 3), 57.5% (day 7), and 61.2% (day 14), respectively, vs WT+UUO+Veh group  T lymphocyte was located around vessels in the kidneys from WT+UUO+Veh animals (data not shown). To identify the subtype of CD3+ T lymphocytes, we preformed immunohistostaining of CD4 and CD8. The data showed that the infiltrating T lymphocytes in UUO groups were identified as CD4-positive stained (CD4+) , but not2AR inactivation significantly decreased this ratio by 54.8% in KO+UUO+Veh group, compared with WT+UUO+Veh group  at day 14 post-UUO. Together, these findings suggest that CD4+ T lymphocyte was the major component in the inflammatory infiltration after UUO while A2AR-activation suppressed CD4+ T lymphocyte infiltration and enhanced the proportion of Treg cells.Lastly, we performed immunohistochemistry staining of Foxp3 (a marker of T cell), to evaluate the involvement of CD4+CD25+Foxp3+ regulatory (Treg) cells that are important inflammation regulators. We demonstrated the presence of Treg cells in all UUO groups at day 14 . Further2AR activation can protect and postpone RIF in experimental UUO animals by the following findings: (i) A2AR activation significantly attenuated UUO-induced pathology consequence and collagen deposition at early stage post-UUO; (ii) A2AR activation inhibited changes of E-cadherin and SMA – two EMT-related changes in RIF; (iii) A2AR activation attenuated the expression of profibrotic mediators TGF-β1 and its downstream Roh/ROCK1 pathway; (iv) Importantly, those effects were associated with A2AR-mediated suppression on infiltration of T lymphocyte. Conversely, inactivation of A2AR conducted an opposite effect in the above phenotypes. These findings demonstrated that activation of A2AR is of importance in phenotypic conversion of RIF, suggesting A2AR may become a potential therapeutic target against RIF.Our study demonstrate, for the first time, that A2AR manipulation in UUO-induced RIF in mice. The infiltration of lymphocyte, as a macrophage-independent response, plays an important role in the process of RIF and nephritis 2AR, as a Gs coupling protein receptor, can significantly increase cAMP level in immune cells, and in turn, alter immune responses including antigen presentation, T cell activation, clonal expansion, and the survival of immune memory 2AR significantly reduced the CD4+ T cell infiltration whereas genetic inactivation of A2AR exerted the opposite effect. Noteworthy, while macrophages played an important role in renal fibrosis in a model of immune-associated chronic inflammation Regulation of the infiltration of T lymphocytes is the principal mechanism of A2AR activation whereas deletion of A2AR abolished Tregs' regulatory effect 2AR activation by CGS21680 significantly increased the ratio of Tregs to CD4+ T lymphocytes, whereas this ratio was significantly decreased in A2AR KO mutants post-UUO. Thus, regulation of Tregs recruitment and (CD4+) T lymphocyte infiltration acts as underlying mechanism of A2AR-mediated effects against RIF.Treg cells are critical to maintain immune-cell homeostasis by enforcing a dominant negative regulation on other immune cells. Thus, Tregs are of great interest due to its immunosuppressive effect and inhibitory effect against fibrosis 2AR could affect EMT-related changes in E-cadherin and SMA. While more direct evidence and evaluations are needed in human studies, EMT is recently proposed as a crucial mechanism in RIF 2AR restored expression level of α-SMA and E-cadherin to a basal level in sham animals  act as a mitogenic factor to affect collagen synthesis and (ii) facilitate the EMT process 2AR restored both aforementioned consequences of TGF-β1 post-UUO. Furthermore, this study showed that TGF-β1-mediated EMT is regulated by the Rho/ROCK-dependent signaling pathway 2AR may, via ROCK1, regulate cell adhesion of tubular epithelial cells and the EMT process. Further evidence is needed to address this potential mechanism.Another important finding in this study is that A animals . Though 2AR after UUO may account for a compensatory protective mechanism. In line with our finding, Lee et al also demonstrated this phenomena 2AR mRNA attribute to inherent renal cells or immigrated cells, e.g., bone marrow-derived cells 2AR-mediated protection against RIF. Noteworthy, some of the A2AR activation resulting effects on post-UUO animals were blunt in the late stage after UUO with unknown reason. This may be due to the severity of phenotypes in late post-UUO stage and the progressive aggravation in pathology unless the pathogenic factors of tubular obstruction might have been removed. The severe pathology changes in late post-UUO stage might be irreversible; however, we also noted the suppressive effect of A2AR activation on expression of TGF-β1 was devoid at day 14 after UUO. This may be due to a redundant mechanism between ROCK1 and TGF-β1, for instance a down-regulation of ROCK1 by A2AR agonist may result in an increased expression of TGF-β1 The increase of A2AR activation in preventing the progression of RIF in the UUO animal model. This provides a novel therapeutic strategy against renal interstitial fibrosis by targeting the adenosine A2A receptor.In summary, this study for the first time demonstrates the beneficial effect of A


A protective antibody epitope was located in the stalk region of the surface glycoprotein, hemagglutinin, and a CD4+ T cell epitope was located in the fusion peptide of the hemagglutinin. The fusion peptide was also found to contain antibody epitopes in humans and animals. A short stretch of well-conserved peptide was also identified in the other surface glycoprotein, neuraminidase, and antibodies binding to this peptide were generated by peptide immunization in rabbits. Although PB1, the only protein which has relatively high overall sequence homology between influenza A and B viruses, is not considered an immunodominant protein in the T cell responses to influenza A virus infection, amino acid sequence comparisons show that a considerable number of previously identified T cell epitopes in the PB1 of influenza A viruses are conserved in the PB1 of influenza B viruses. These data indicate that B and T cell cross-reactivity exists between influenza A and B viruses, which may have modulatory effects on the disease process and recovery. Although the antibody titers and the specific T cell frequencies induced by natural infection or standard vaccination may not be high enough to provide cross protection in humans, it might be possible to develop immunization strategies to induce these cross-reactive responses more efficiently.Influenza A and B viruses form different genera, which were originally distinguished by antigenic differences in their nucleoproteins and matrix 1 proteins. Cross-protection between these two genera has not been observed in animal experiments, which is consistent with the low homology in viral proteins common to both viruses except for one of three polymerase proteins, polymerase basic 1 (PB1). Recently, however, antibody and CD4 Causative agents, influenza A and B viruses, belong to the family Orthomyxoviridae[Human influenza is a highly contagious acute respiratory illness that is responsible for significant morbidity and excess mortality especially in the elderly and the very young worldwide. On average 5% to 20% of the population in the United States acquires influenza every year with more than 200,000 people hospitalized from influenza complications, while influenza-related deaths range from 3000 to 49,000  induced peptide specific antibodies in rabbits, but these antibodies (antisera) were not neutralizing. Peptide immunization (with complete Freund’s adjuvant) did not protect mice from infection with either influenza A or B viruses ), although monoclonal antibodies established from immunized mice were neutralizing in vitro (the antisera were not) [Although the overall homology between the HAs of influenza A and B viruses is only 18% (Table ere not) .More than 20 years later there is renewed interest in the fusion peptide as a target of antibodies ,30-32. C50) [50) [Stanekova et al. showed that peptide immunization with the fusion peptide (the first 38 amino acids of HA2 domain of influenza A/Mississippi/1/85(H3N2) strain conjugated to the KLH mixed with Freund’s adjuvant) produced anti-fusion peptide antibodies and protected mice against mild challenge with homologous and heterologous (H1N1 or H7N1) viruses (1 and 2 LD50) ,33. Prab50) [50) . Liu et 50) [50) .50). Hybridomas from the former immunization did produce the fusion-peptide specific antibodies which were neutralizing in vitro [In these four mouse experiments BALB/c mice were used for immunization, suggesting that anti-fusion peptide antibody production may depend on immunization strategy. Immunization with purified whole HA protein induced a neutralizing anti-fusion peptide antibody . Immunizin vitro . In contin vitro .Liu et al. also reported that they did not detect antibodies binding to GLFGAIAGFIE peptide in the sera of adult humans, which is different from the report by Atassi and Webster , althoug+ T cell epitope which was also located in the fusion peptide when we were analyzing T cell responses to the H2 HA [+ T cell epitope (RG(L/I/F)FGAIAGF(I/L)E(G/N)G)a in the fusion peptide. Since the fusion peptide is highly conserved in both influenza A and B viruses, we decided to test the cross-reactivity of a CD4+ T cell line generated from one of the donors’ PBMCs. As expected the T cell line secreted interferon-γ and tumor necrosis factor-α after stimulation with influenza A/H1N1 , A/H2N1, A/H3N2, A/H5N1 and B viruses and lysed target cells infected with these viruses or pulsed with the purified HA proteins. The epitope is likely to be restricted by the HLA-DR  [+ T cells can control influenza virus infection by producing inflammatory cytokines and chemokines, directly lysing infected cells, and by helping B cells and CD8+ T cells [We identified a CD4he H2 HA . We dete allele) . CD4+ T  T cells ,37. Wilk T cells .+ T cell epitope was also identified in the fusion peptide. Gianfrani et al. identified peptides which had an HLA-A2.1 binding motif and bound to the HLA-A2.1 molecule (GLFGAIAGFI) within the fusion peptide based on the sequence of influenza virus A/Puerto Rico/8/34(H1N1) strain [+ T cells in humans proving that the peptide is indeed the HLA-A2.1-restricted CD8+ T cell epitope [+ T cell epitope, it may not be cross-reactive to influenza B viruses when we consider the amino acid change from leucine/isoleucine to phenylalanine at one of the anchor residues (2nd position).A CD8) strain . This HL epitope . AlthougA protective antibody epitope cross-reactive to influenza A and B viruses was also located in the stalk region of the HA. Dreyfus et al. cloned human monoclonal antibodies from B cells from volunteers vaccinated with the seasonal influenza vaccine and one of these antibodies, CR9114, bound to both influenza A and B viruses tested. Although the monoclonal antibody neutralized only influenza A viruses in vitro, it protected mice against both influenza A and B viruses challenge. This monoclonal antibody was found to bind to the stalk (stem) region of the HA .The stalk-specific antibodies tend to have broader neutralizing activity than the conventional globular head-specific antibodies among influenza A viruses and are being studied extensively (reviewed in ) includiIt is interesting and important to know if these immunization strategies can also induce the fusion peptide-specific antibodies.Some antibodies against the NA can bind near its catalytic site and inhibit virus release from infected cells and these epitopes are conformational . A short+ and CD8+ T cell epitopes than any other influenza A virus proteins in humans [+ T cell epitopes restricted by HLA-A1, -A2, -A3, -A24, -B7 and -B44 supertypes which are highly conserved among H1N1 , H2N2, H3N2, H5N1, H7N7 and H9N2 subtypes, six epitopes are on PB1. Only nucleoprotein has more epitopes (eight) [Among influenza A and B viral proteins only polymerase basic 1 has relatively high overall sequence homology compared to other viral proteins (Table n humans . Of 25 C (eight) .50) [+ and CD8+) may have been induced in vaccinated mice and contributed to the observed protection.In mice, a DNA vaccine expressing the PB1 protein from A/Puerto Rico/8/34(H1N1) strain protected against homologous challenge  . Anti-PB50) , it is p+ T cell epitopes and two were CD4+ T cell epitopes. Most of the CD8+ T cell epitopes are restricted by HLA-A1, -A2, -A3 or -A24 supertypes [+ T cell epitopes are restricted by HLA-DR supertype [+ and CD4+ T cell epitopes are expected to be recognized by a majority of human population. Eleven of the 14 conserved T cell epitopes were clustered in two regions, amino acid 404–422 and 471–517. The same regions also contain several less-characterized T cell epitopes . The presence of these epitopes suggests that there are cross-reactive T cell responses between the PB1 proteins of influenza A and B viruses.We looked at amino acid sequence conservation in T cell epitopes in the PB1 reported in the literature . The result is summarized in Table pertypes  and the upertype  suggesti+ T cell responses [+ and CD8+ T cell responses.Commercial TIVs do not contain detectable levels of PB1 by two-dimensional high-performance liquid chromatography ,67, and esponses . It is n+ and CD8+ T cell responses tend to be more subtype-cross-reactive than antibody responses [+ and CD8+ T cell responses against influenza viral proteins  (4 LD50), although the relative contribution of CD4+ T cell responses to the PB1 epitopes in protection was not known [Since CD4esponses ,25,69, tiewed in , and thec groups . In the ot known .The data we reviewed here show that B and T cell cross-reactivity exists between influenza A and B viruses, which may have modulatory effects on the disease process and recovery. Although the antibody titers and the specific T cell frequencies induced by natural infection or standard vaccination may not be high enough to provide cross protection, it might be possible to develop immunization strategies to induce these cross-reactive responses more efficiently. Specifically, immunization strategies to induce the stalk-specific antibodies might also be efficacious for induction of the fusion peptide-specific antibodies . DNA vaccine or recombinant viral vectors expressing PB1 cDNA alone may not be protective against high dose challenge, but may enhance protection against influenza conferred by immune responses to HA or NA.aThe conserved sequence reported in the paper [he paper  containePB1: Polymerase basic 1; NP: Nucleoprotein; M1: Matrix 1 protein; HA: Hemagglutinin; NA: Neuraminidase; NS1: Nonstructural protein 1; NEP: Nuclear export protein; M2: Matrix 2 protein; PB2: Polymerase basic 2; PA: Polymerase acid; LD50: 50% lethal dose; KLH: Keyhole limpet hemocyanin.The authors declare that they have no competing interests.MT wrote the initial draft. All authors discussed, read and approved the final manuscript.


The clonal human neuroblastoma BE(2)-M17 cell line is known to differentiate into a more prominent neuronal cell type by treatment with trans-retinoic acid. However, there is a lack of information on the morphological and functional aspects of these differentiated cells.Immortalized neuronal cell lines can be induced to differentiate into more mature neurons by adding specific compounds or growth factors to the culture medium. This property makes neuronal cell lines attractive as 2+) channels and (c) Ca2+-dependent neurotransmitter ([3H] glycine) release in cultured BE(2)-M17 cells. Cells treated with 10 μM trans-retinoic acid (RA) for 72 hrs exhibited marked changes in morphology to include neurite extensions; presence of P/Q, N and T-type voltage-gated Ca2+ channels; and expression of neuron specific enolase (NSE), synaptosomal-associated protein 25 (SNAP-25), nicotinic acetylcholine receptor α7 (nAChR-α7) and other neuronal markers. Moreover, retinoic acid treated cells had a significant increase in evoked Ca2+-dependent neurotransmitter release capacity. In toxicity studies of the toxic gas, phosgene (CG), that differentiation of M17 cells with RA was required to see the changes in intracellular free Ca2+ concentrations following exposure to CG.We studied the effects of trans-retinoic acid treatment on (a) some differentiation marker proteins, (b) types of voltage-gated calcium -M17 cells may serve as a better neuronal model to study neurobiology and/or neurotoxicity. In our and other laboratories, Ca2+-dependent [3H] glycine release inhibition due neurotoxins (e.g. botulinum neurotoxins and tetanus toxin) has been shown to be a sensitive indicator of toxicity in neuronal models such as cultured primary mouse spinal cord cells  glycine for 30 min at 37°C to label a releasable [3H] glycine pool. Cells were then washed with a series of low K+ (3 mM)-containing isotonic buffers . Unless otherwise indicated, [3H] glycine release was stimulated by addition of 80 mM K+ and 2 mM Ca2+ to cultures; stimulation medium was collected after 5 min at 37°C. Calcium-dependent release was determined by subtracting baseline radioactivity secreted from cultures in the absence of Ca2+, and expressed as a percentage of the total cellular radioactivity.High potassium-stimulated Caescribed . Culture3H]-Valine  for 24 hours. The medium was then aspirated and cells were washed two times with 1 ml of a physiological balanced solution (PBS) containing in mM: 128 NaCl, 5.9 KCl, 1.28 CaCl2, 1.2 MgCl2, 17 HEPES, 3.3 glucose. All components for this solution were obtained from Sigma-Aldrich . Cells were then incubated with an additional 1 ml aliquot of PBS for one hour for equilibration. The PBS was replaced with either PBS or stimulation medium containing 25 – 100 mM KCl and 1 μCi/ml 45CaCl2. The different KCl concentrations were adjusted by reducing the NaCl concentration proportionately to maintain iso osmolality. The addition of 1 μCi/ml of 45CaCl2 did not significantly alter the osmolality or the concentration of CaCl2 (data not shown). After 4 minutes, the radioactive medium was aspirated and ice-cold PBS was used to wash cells twice. The cells were then lyzed by adding 0.4 N NaOH solution and rocked overnight at 4°C. The next day, the 0.4N NaOH solution was pH neutralized with 0.4N HCl + Tris. The neutralized solution was added to scintillation fluid and 45Ca and 3H levels were measured on a Beckman LS6500 multi-purpose scintillation counter . Calcium-dependent release was determined by subtracting baseline radioactivity secreted from cultures in the absence of Ca2+, and expressed as a percentage of the total cellular radioactivity. Total cellular radioactivity was calculated by adding the 45Ca2+ cpm released upon stimulation with high KCl and the 45Ca2+ cpm present in the resting medium with low KCl before and after stimulation.M17 cells were seeded into 24 well plates  and cultured with 1 μCi/ml of  glycine) release described below were either not prominent (morphology) or inconsistent  (data not shown). The treatment conditions that we selected were previously used to show early effects of RA-induced neuronal differentiation and oncogene (p26-Bcl-2) expression in another BE(2) variant cell clone i) in both undifferentiated and RA differentiated M17 cells. First, we tested the effect of the non-specific Ca2+ ionophore A23187 (5 μM) on [Ca2+]i. The results shown in Figure 2+]i due to A23187 as expect without any significant difference between undifferentiated vs. differentiated cells. CG (16 ppm) caused significant decreases (p<0.05) at all times tested only in the RA differentiated cells without any effect in undifferentiated cells  primary cell cultures, (2) clonal cell lines, and (3) neural stem cells. The main advantage of using primary cell cultures is that they retain the morphological, neurochemical, and electrophysiological properties of neurons in situi: Intracellular free CaThe authors declare that they have no competing interests, financial or non-financial.Participated in research design: DA, BK, KH, PMcNutt, RR. Conducted experiments: DA, BK, JP, BB, KH. Contributed new reagents or analytic tools: N/A. Performed data analysis: DA, BK, JP, KH. Wrote or contributed to the writing of the manuscript: DA, BK, KH, PMcNutt, RR. Other: DA, BK, RR acquired funding for the research. All authors read and approved the final manuscript.Split confocal image of synapsin-1/2 and β3-tubulin expression in RA-induced M17 cells at 120 h. M17 neuroblastoma cells were grown on cover slips. Cells were fixed, stained, and immunofluorescent images were taken (63X). Synapsin-1/2 (green), β3-tubulin (red) and nuclei (blue). Split panels diffuse synapsin expression in cell body; with punctuate expression apparent in elongated neurites.Click here for file


SLC26A4, which encodes pendrin, are a common cause of deafness. SLC26A4 mutations are responsible for Pendred syndrome and non-syndromic enlarged vestibular aqueduct (EVA). The mutation spectrum of SLC26A4 varies widely among ethnic groups. To investigate the incidence of EVA in Chinese population and to provide appropriate genetic testing and counseling to patients with SLC26A4 variants, we conducted a large-scale molecular epidemiological survey of SLC26A4.Mutations in SLC26A4, exons 8, 10 and 19 were sequenced. For patients with one allelic variant in the hot spot regions, the other exons were sequenced one by one until two mutant alleles had been identified. Patients with SLC26A4 variants were then examined by temporal bone computed tomography scan for radiological diagnosis of EVA. Ten SLC26A4 variants were cloned for functional study. Confocal microscopy and radioisotope techniques were used to examine the membrane expression of pendrin and transporter function.A total of 2352 unrelated non-syndromic hearing loss patients from 27 different regions of China were included. Hot spot regions of SLC26A4 are distinct among both ethnicities and regions of Mainland China. Most of the variants caused retention of pendrin in the intracellular region. All the mutant pendrins showed significantly reduced transport capability.Of the 86 types of variants found, 47 have never been reported. The ratio of EVA in the Chinese deaf population was at least 11%, and that in patients of Han ethnicity reached at least 13%. The mutational spectrum and mutation detection rate of SLC26A4 in China is provided. The functional assessment procedure can be applied to identification of pathogenicity of variants. These findings are valuable for genetic diagnosis, genetic counseling, prenatal testing and pre-implantation diagnosis in EVA families.An overall description of the molecular epidemiological findings of  SLC26A4 encodes an anion transporter transmembrane protein, pendrin, which is expressed in the thyroid, kidney, and cochlea -, HCO3-, OH-, I-, or formate −/HCO3- exchange in the acid–base regulating B- and non-A–non-B-intercalated cells −/HCO3- exchange, and is therefore involved in the conditioning of endolymphatic fluid, presumably due to HCO3- secretion SLC26A4 resulting in hearing loss, enlargement of the vestibular aqueduct (EVA) and iodine organification defect in the thyroid gland SLC26A4 mutations on the development of goiter, the expression of goiter in PS patients is variable and may have incomplete penetrance Pendred syndrome (PS) is the most common form of syndromic deafness and accounts for about 10% of hereditary hearing impairment SLC26A4 mutations have been reported SLC26A4, consistent with autosomal recessive disorder, whereas patients with non-syndromic hearing loss and EVA might have one or no mutant allele SLC26A4 are 57% and 81%, respectively SLC26A4 allele detected To-date, 174 SLC26A4 mutations are the second-most common cause of deafness in China. The incidence of non-syndromic EVA accounts for 13.73% of hereditary hearing loss SLC26A4 variants in the Chinese hearing loss population, of which at least half are novel. With genetic testing of deafness becoming more popular in China, verification of the pathogenicity of novel variants is essential for both genetic and prenatal diagnosis. However, the two parameters used thus far–(i) low incidence of the mutation in the control population and (ii) substitution of evolutionarily conserved amino acids by the mutation–are not sufficient for clinical genetic diagnosis. To investigate the incidence of EVA in Chinese hearing loss populations and provide appropriate genetic testing and counseling to patients with SLC26A4 variants, we conducted a large-scale molecular epidemiological survey of SLC26A4 in China. In addition, in order to assess the pathogenicity and understand the molecular mechanism(s) underlying EVA and hearing loss, we examined the membrane expression of pendrin and the transporter function of 10 variants that comprised six novel variants and four reported only in Chinese patients.A total of 2352 unrelated non-syndromic hearing loss patients from 27 different regions of China were included in this study. To sample as many regions of China as possible, we included the remote northwestern provinces, including Xinjiang (translates as ‘New Territory’), Tibet, and Qinghai, where minorities make up a significant proportion of the local population. We also included samples from the southwestern provinces of Yunnan and Guizhou, where the populations comprise a number of minorities originating from various native tribes. The patients consisted of 1314 males and 1038 females ranging in age from 1.8 to 24 years with an average age of 14.17±3.41. The majority of patients were Han Chinese (1903), followed by minorities in the Southwest region (125), Tibetan (119), Hui (89), minorities in Xinjiang (69), Mongolian (33), Maan (12) and Korean (2). Ethnic subgroup designations were based on permanent residency documentation.The subjects in this study were from special educational schools within each region. All patients showed moderate to profound bilateral sensorineural hearing impairment on audiograms. Careful medical examinations revealed no clinical features other than hearing impairment. In addition to the 2352 patients, 200 Han individuals with normal hearing  were recruited as control subjects from Beijing (Northern) and Jiangsu Province (Eastern), two densely populated regions consisting of 98 to 99% Han Chinese. Auditory function was evaluated by pure-tone air-and bone-conduction threshold audiometry.Written informed consent was obtained from all subjects or guardians prior to blood sampling and genetic testing. The study protocol including the consent procedure was performed with the approval of the Ethics Committee of the Chinese PLA General Hospital.SLC26A4 includes 2–20 exons. DNA sequence analyses of SLC26A4 were performed by polymerase chain reaction (PCR) amplification of the coding exons plus approximately 50–100 bp of the flanking intron regions followed by Big Dye sequencing and analysis using an ABI 3100 DNA sequencing machine  and ABI 3100 Analysis Software v.3.7 NT, according to the manufacturer’s instructions. Because about 90% of Chinese EVA patients carried at least one allelic mutation in exons 8, 19 or 10 of SLC26A4SLC26A4 were sequenced one by one until two mutant alleles had been identified.DNA was extracted from peripheral blood leukocytes using a commercially available DNA extraction kit . The open reading frame (ORF) of SLC26A4 for mutations or variants by DHPLC followed by sequencing analysis.Normal-hearing controls were screened in exons 2–21 of SLC26A4 were examined by temporal bone CT scan for diagnosis of inner ear malformation or EVA based on a diameter of >1.5 mm at the midpoint between the common crus and the external aperture. Some patients with EVA were further examined using magnetic resonance imaging (MRI).Patients with mutations or variants in XhoI and KpnI restriction sites. The pendrin cDNA sequence was verified by nucleotide sequencing, and was identical to a registered sequence (GenBank NM_000441). Oligonucleotide directed mutagenesis was performed to create constructs of 10 mutant plasmids using the QuikChange® Site-Directed Mutagenesis Kit , according to the manufacturer’s protocol. The cloning and mutagenic primers used in this study are listed in The coding region of pendrin cDNA was amplified by PCR from the cDNA library of human thyroid tissue and subcloned into the pEGFP-N1 vector  using the 5 cells per well. Cells were quantified using a hemocytometer . The quantity of DNA used per well was 0.8 µg, added to 1.6 µl lipofectamine. Fluorescence microscopy was used to evaluate membrane targeting during cell-selection experiments.Human embryonic kidney (HEK) cells were cultured in DMEM solution  supplemented with 10% fetal bovine serum. Constructs of pendrin and its mutated plasmids were introduced to the cells using lipofectamine 2000 . The amount of DNA used per 35-mm dish was 4 µg, mixed with 10-µl lipofectamine. For radioisotope uptake experiments, the cells were passaged into 24-well plates 24-h before transfection, at 2×1014C] formate uptake, sorted cells in the 24-well culture cluster were first incubated for 30 min in a solution containing 130 mM NaCl, 20 mM HEPES, 5 mM KCl, 5 mM glucose, 2 mM CaCl2 and 1 mM MgCl2 (pH 7.3 and 305 Osm/L). Cells were then incubated at RT for 12 min in a solution containing 140 mM K-gluconate, 20 mM HEPES and 5 mM glucose (pH 7.3 and 305 Osm/L). [14C] formate  was added to this solution at a concentration of 20 µM. Cells were then washed three times with cold K-gluconate solution without [14C] formate, lysed with 200 µl 0.5 M NaOH, and neutralized with 0.5 M HCl. The lysate was used for liquid scintillation counting to determine the [14C] formate uptake. In each run, three wells were assayed for each plasmid, and the experiments were repeated three times independently. Therefore, the data in each group represented a sample size (n) of 3×3 = 9 trials for each plasmid and control. Student’s t-test was used for statistical analysis.Conventional radioisotope techniques were used to measure transport functions from HEK cells transfected with plasmids encoding for pendrin and its mutated products, as described in previous studies SLC26A4 in 2352 patients, we identified 342 patients with mutations or variants, of which 116 were homozygotes and 226 were heterozygotes. For the 226 patients with monoallelic mutations or variants in exons 8, 10, or 19 of SLC26A4, 132 were verified as compound heterozygotes by the subsequent whole ORF analysis, and the other 94 were verified as heterozygotes. The total variant detection rate was 14.54% (342/2352), including a homozygous mutation detection rate of 4.93% (116/2352), a compound heterozygous mutation detection rate of 5.44% (128/2352), and a monoallelic mutation detection rate of 4.17% (98/2352). For patients of Han ethnicity, accounting for 80.52% of our sample, the total variant detection rate, homozygous mutation detection rate, compound heterozygous mutation detection rate and monoallelic mutation detection rates were 16.55% (315/1903), 5.68% (108/1903), 6.46% (123/1903) and 4.41% (84/1903), respectively. Eighty-six types of variants in SLC26A4 were found, including 7 nonsense variants, 13 frameshift variants, 48 missense variants, 5 splicing site variants, 6 silent variants, 1 untranslated region (UTR) variant and 6 intron variants  and a mutant allele frequency of 61.6% (377/612). p.H723R was the second-most frequent hot-spot mutation in Han, Hui, and Mongolian ethnicities with an allele frequency of 1.51% (71/4704) and a mutant allele frequency of 11.6% (71/612). In contrast, neither IVS7-2A>G nor p.H723R were found in the Tibetan and Uygur patients.The mutational spectrum of The mutation detection rates were distinct in the hearing loss populations of different ethnicities, with the highest detection rate in the Han population (16.55%), followed by the Hui population  and the Mongolian population . In contrast, the mutation detection rate in the Tibetan population, and minorities in the Southwest region and Uygur, were low at 4.2, 2.4 and 1.45%, respectively.SLC26A4 mutation detection rates differed geographically in China. Henan province ranked first with a detection rate of 31.58%. Detection rates in Gansu, Heibei, Hubei, Shaanxi and Heilongjiang province were 26.32, 25, 18.75, 18.37 and 16.67%, respectively. The SLC26A4 mutation detection rate in Guangxi province, the Tibet Autonomous Region and Guizhou province were the lowest at 3.3, 5.08 and 5.4%, respectively. In Guangxi province, no biallelic SLC26A4 mutations were found in the 90 patients enrolled. In the Tibet Autonomous Region, biallelic SLC26A4 mutations were detected only in patients of Han ethnicity. The geographic distribution and the proportion of patients carrying two SLC26A4 mutant alleles in each region are shown in SLC26A4 genetic testing or by high resolute temporal bone CT scan.The SLC26A4 mutations in China is estimated to be about 2.5%. Polymorphisms in SLC26A4 appear to be rare in the general population in comparison to those in GJB2Four IVS7-2A>G heterozygotes, one missense c.2176A>G (p.I726V) heterozygote, one silent variant c.2217A>G (p.Q739Q), one silent variant c.225C>G (p.L75L) and one intron variant (intron4-12T>A) were found in the control group. Although this control population may be too small to reach a final conclusion, the carrier rate of SLC26A4, 320 were examined by temporal CT scan or MRI. The follow-up of the remaining 22 patients  failed. All patients (240/240) with biallelic mutations or biallelic variants (not including silent variants) and 23.75% (19/80) of those with monoallelic mutations or variants in the ORF of SLC26A4 were confirmed to have EVA by temporal bone CT scan or MRI , c.665G>T (p.G222V), c.941C>T (p.S314L), c.1517T>G (p.L506R), c.1985G>A (p.C662Y), and c.2326C>G (p.R776G) have not been reported before. c.259G>T (p.D87Y), c.1079C>T (p.A360V), c.1225C>T (p.R409C) and c.1991C>T (p.A664V) have only been detected in ethnic Chinese SLC26A4 orthologs. However, L506, C662 and A664 are located in conserved regions of most of the SLC26A4 orthologs  in HEK cells after transfecting the cells with plasmids containing one of the 10 variants tagged by EGFP. Di-8-ANEPPS is a member of the ANEP (aminoaphthyl ethenyl pyridinium) class of membrane potential dyes. Co-localization of EGFP expression and the di-8-ANEPPS dye in the membrane would suggest proper membrane targeting of the proteins 3- exchange activities at the apical membrane of inner ear epithelial cells is the key factor that causes EVA and deafness 14C] formate uptake after sorting of EGFP-positive cells by fluorescence-based flow cytometry. Cells transfected with EGFP-vector only were used as a negative control. As shown in P<0.01). Pfarr et al. reported that the p.R776C mutation does not compromise transporter activity It has been suggested that the defect in Cl−/HCOSLC26A4, which encodes pendrin, are a common cause of deafness, and are responsible for both syndromic and non-syndromic hearing loss. The mutation spectrum of SLC26A4 varies widely among ethnic groups Mutations in SLC26A4 gene in hearing loss populations have been reported. Previous studies verified that about 90% of Chinese patients with EVA carried at least one allelic mutation in exons 8, 19 or 10 of SLC26A4SLC26A4 is a large gene with 20 exons, hot-spot region screening in a molecular epidemiological survey is an economical method. SLC26A4 mutations were detected in nearly 15% of 2352 non-syndromic Chinese hearing impairment patients, with IVS7-2A>G being the most prevalent. By screening the above three exons of SLC26A4, we verified that the ratio of EVA in the Chinese deaf population was at least 11%, and that in the Han ethnicity reached at least 13%. Of our patients with EVA, 23.75% carried only one missense mutation. Whether the missense mutation causes a dominant negative effect and/or specifies a different phenotype is not clear. It is possible that the second mutant allele has not yet been identified due to the location of mutations deep in introns or promoter regions that were not sequenced, intragenic exon deletions, or the involvement of mutations in genes other than SLC26A4, such as FOXI and KCNJ10 . However, none of our patients with one SLC26A4 missense mutation carried any of the reported FOXI or KCNJ10 mutations.All of the above studies focused on patients with EVA or Pendred syndrome. Few large-scale molecular epidemiological surveys on the SLC26A4 mutational spectra and detection rates, which may be explained by the genetic heterogeneity in various ethnicities. For example, the mutation detection rates in the Tibetan population and minorities in the Southwest region and Uygur populations were significantly lower than that in the Han, Hui and Mongolian populations. Although IVS7-2A>G and p.H723R were hot mutations in Han, Hui and Mongolian patients, they were not found in the Tibetan and Uygur patients. Our results also revealed that the SLC26A4 detection rate and incidence of EVA varied geographically. The incidence of inner ear malformation in the Tibetans living in the Tibet plateau was almost as high as that in Chinese Han patients, but the types of malformation in the Tibetan population differed greatly GJB2, was significantly lower in Guangxi province (6.05%) than the average rate in China (21.01%) We found that different ethnicities that originated from various regions of China showed distinct SLC26A4 mutations prior to temporal bone CT scan for identifying patients with EVA. In China, the cost of a temporal CT scan is 200 to 300 RMB . Because of the relatively high cost, it is not possible to perform a CT scan in every hearing loss patient for diagnosis of EVA. Because 97.9% of Chinese patients with EVA carry an SLC26A4 mutation SLC26A4 mutations in hearing loss patients indicate a high possibility of EVA. This model has the unique advantage of identifying patients with EVA in an epidemiologic study in large-scale deaf populations.A striking achievement of this study is the establishment of a new strategy for detecting SLC26A4. Functional tests of mutated pendrin allelic variants found in patients with Pendred syndrome or non-syndromic EVA revealed that the pathology is linked to a reduction or a loss of function in the ion transport activity of pendrin The discovery of 47 novel variants enriches the mutational spectrum of We used confocal microscopy and radioisotope techniques to examine the membrane expression and transporter function of 10 mutant proteins in human embryonic kidney cells. We showed that all 10 mutations resulted in a significant reduction in membrane expression of the proteins. Measurement using a radioisotope technique showed that their transport activity was significantly reduced. Our results suggest that the pathogenesis underlying the hearing loss associated with these mutations is the result of reduced membrane expression and decreased transport capability of the proteins.SLC26A4 sequence is detrimental to its transport function. However, Pfarr et al. SLC26A4 (a 780 amino acid protein). It is thought that the N- and C-termini of a protein are not structural and the mutations occurring in these areas probably have little or no functional impact Pera et al. SLC26A4 in China. We showed that the prevalence of EVA in the Chinese deaf population was at least 11%, which reached at least 13% in the Han majority population. The mutational spectrum and mutation detection rate of SLC26A4 are distinct among both ethnicities and regions of mainland China. Our results suggest that the pathogenesis underlying hearing loss in patients with EVA is the result of reduced membrane expression and the decreased transport activity of the mutant pendrins. The functional assessment procedure can be applied to genetic diagnosis of EVA patients and prenatal diagnosis for their families. These findings are valuable for genetic counseling and risk assessment for families with EVA in China.In conclusion, we provide an overview of the molecular epidemiological status of Figure S1Geographic distribution and the proportion of patients carrying two SLC26A4 mutant alleles in each region studied. NE: not examined(TIF)Click here for additional data file.Figure S2Examples of CT and MRI images of normal children and children with SLC26A4 mutations. A: CT of temporal bone of a normal child. Arrows show the normal vestibular aquaduct. B: CT of a patient with EVA. Arrows mark the enlarged vestibular aquaduct. C: MRI of child with normal inner ear. Arrows indicate the normal endolymphatic sac. D: MRI of a patient with EVA. Arrows mark the hydrop of endolymphatic sac.(TIF)Click here for additional data file.Figure S3Representative pure-tone audiograms of children with SLC26A4 mutations. A: c.259G>T/c.2168A>G. B: c.941C>T/c.1174A>G. C: c.1517T>G/IVS7-2A>G. D: c.1991C>T/IVS7-2A>G(TIF)Click here for additional data file.Figure S4Protein sequences of 13 SLC26A4 orthologs, including: (1) Homo sapiens (NP_000432.1), (2) Nomascus leucogenys (XP_003268184.1), (3) Pan troglodytes (XP_519308.2), (4) Macaca mulatta (XP_001094049.1), (5) Callithrix jacchus (XP_002751785.1), (6) Sus scrofa (XP_003357559.1), (7) Canis lupus familiaris (XP_540382.3), (8) Rattus norvegicus (NP_062087.1), (9) Mus musculus (NP_035997.1), (10) Oryctolagus cuniculus (XP_002712085.1), (11) Loxodonta africana (XP_003407255.1), (12) Xenopus (Silurana) tropicalis (NP_001107135.1), and (13) Monodelphis domestica (XP_001363598.1).(TIF)Click here for additional data file.Figure S5Heterologous expression of wild-type and mutant pendrins. HEK cells were transfected with each plasmid of the 10 variants tagged by EGFP. A membrane-based dye (orange color), di-8-ANEPPS, was also added in the solution before confocal microscopy. Co-localization of EGFP expression and the di-8-ANEPPS dye in the membrane would suggest proper membrane targeting of the proteins.(TIF)Click here for additional data file.Figure S6Transport activity of wild-type pendrin and 10 mutant pendrins.(TIF)Click here for additional data file.Methods S1SLC26A4 Site-Directed Mutagenesis Primers. The mRNA from normal human adenoid specimens was extracted, and cDNA was synthesized. PCR was done to catch the target gene. Primers were as follows: XhoI -F:5′- CCGCTCGAGATGGCAGCGCCAGGCGGCAG -3′ KpnI -R:5′- GGGGTACCGTGGATGCAAGTGTACGCATAG -3′XhoI -F contains protective base and XhoI restriction sites, its 5 'end partial sequence for PCR amplify target gene. KpnI -R contains protective base and KpnI restriction sites,its 3 'end partial sequence for PCR amplify target gene. The entire ORF of the human SLC26A4 gene was cloned into the expression vector pEGFP-N1 . The point mutation-specific primers were synthesized as shown in (DOCX)Click here for additional data file.Table S1SLC26A4 Site-Directed Mutation Primers.(DOCX)Click here for additional data file.


A one year active surveillance program for influenza A viruses among avian species in a live-bird market (LBM) in Bangkok, Thailand was conducted in 2009. Out of 970 samples collected, influenza A virus subtypes H4N6 (n = 2) and H4N9 (n = 1) were isolated from healthy Muscovy ducks. All three viruses were characterized by whole genome sequencing with subsequent phylogenetic analysis and genetic comparison. Phylogenetic analysis of all eight viral genes showed that the viruses clustered in the Eurasian lineage of influenza A viruses. Genetic analysis showed that H4N6 and H4N9 viruses display low pathogenic avian influenza characteristics. The HA cleavage site and receptor binding sites were conserved and resembled to LPAI viruses. This study is the first to report isolation of H4N6 and H4N9 viruses from birds in LBM in Thailand and shows the genetic diversity of the viruses circulating in the LBM. In addition, co-infection of H4N6 and H4N9 in the same Muscovy duck was observed. Live-bird markets (LBMs) are the places where wild birds, pet birds, meat birds and domestic poultry are sold to households. In Asia including Thailand, due to the cultural preference of consuming freshly slaughtered poultry, LBMs are located in both suburban areas and center of the communities. In the markets, thousands of birds from different sources are sold in wire stacked cages containing densely packed and mixed bird populations. These conditions provide excellent environments for animal to animal and animal to human influenza virus transmissions and may result in an outbreak of influenza A virus in both animals ,2 and huUp to date, many studies on influenza A in LBMs from various countries have been reported. During 2000-2001, 6 subtypes (9 genotypes) of low-pathogenic avian influenza (LPAI) were identified in LBMs in China . Apart fDuring January to December 2009, a 12-month LBM surveillance program was carried out in LBM in Bangkok, Thailand. The LBM was visited monthly and approximately 40 samples (chicken and ducks) per market were collected at each sampling. In total, 970 samples  were collected from 485 animals  from LBM located in the center of Bangkok. All swab samples were subjected to virus isolation by using embryonated egg inoculation according to WHO/OIE recommendations. Allantoic fluid from egg inoculation was tested for Hemagglutination (HA) titer using 1% chicken red blood cells. Samples testing positive for HA test were subjected for influenza A virus identification and subtyping. To identify influenza A virus, real-time-RT-PCR specific for the influenza A virus matrix (M) gene was performed as previously described . To subtCU-LM1973/09(H4N6) (CY062545-52), A/Muscovy Duck/Thailand/CU-LM1983/09(H4N6)(CY062553-60), and A/Muscovy Duck/Thailand/CU-LM1984/09(H4N9)(CY062561-68).After subtyping, whole genome sequencing was performed by using newly designed primers for each gene of influenza A subtypes, H4N6 and H4N9. The whole genome sequences of influenza A virus subtypes H4N6 (n = 2) and H4N9 (n = 1) were submitted to the GenBank database with accession numbers as follows: A/Muscovy Duck/Thailand/Phylogenetic analysis was performed using MEGA 4.1 program  with neighbor-joining method with Kimura 2-parameter. Nucleotide sequences of H4N6 (n = 24) and N9 n = 30) representing different geographic locations, host species and Eurasian/North American lineages were included for phylogenetic analysis. Phylogenetic analysis of the hemagglutinin (HA) gene showed that the H4 gene can be divided into 2 major lineages, the Eurasian and North American, respectively . Sequence homologies of each gene of the H4N6 and H4N9 influenza A viruses with highest nucleotide identities to reference influenza A strains are shown in Table The deduced amino acids of the H4 gene of influenza A isolates were aligned and compared with those of reference H4N6 and H4N9 viruses representing the Eurasian and North American lineages published at the GenBank database using the MegAlign program. Our result showed that amino acids at the HA cleavage site in the Thai H4N6 and H4N9 viruses are "PEKASR", similar to most avian isolates in the Eurasian lineage, while those of the North American lineage including ruddy turnstone (NJ/47/85), mallard (ALB/49/95) and swine ONT/1911-1/99) isolates are "PEKATR" Table 13] The  The 13] 11-1/99 iThe influenza A virus subtypes H4N6 and H4N9 found in LBMs can be a potential risk to birds and humans. The viruses were shed in feces and contaminated the wire stack cages in LBM. This setting provides an appropriate condition for virus transmission among birds kept in the cage nearby or below . ConsequThe public should be made aware of the significance of the influenza viruses, the hosts, the environment and potential virus reassortment especially in LBMs. Examples of influenza prevention and control strategies in the LBM include 1) creating a rest day to interrupt the LBM's flow system, which could significantly reduce the occurrence of the virus in LBMs , 2) disiIn conclusion, influenza A virus subtypes H4N6 and H4N9 were isolated from ducks in a live bird market in Bangkok, Thailand. Both H4N6 and H4N9 subtypes recovered from ducks had never been reported in Thailand. Whole genome sequences of the viruses were determined and submitted to the respective databases. Phylogenetic analysis of H4N6 and H4N9 viruses showed that gene segments of the viruses were derived from the Eurasian linage. Genetic analysis of the viruses also showed that the viruses display low pathogenic characteristics, with amino acids specific to avian influenza viruses.The authors declare that they have no competing interests.TW conducted sample collection, virus isolation, virus identification, genome sequencing, phylogenetic analysis and drafted the manuscript. MW, SB, NB participated in sample collection, virus isolation and whole genome sequencing. PK helped designed LBM surveillance, data analyses. AA designed LBM surveillance, data analyses and final approval of the manuscript. All authors read and approved the final manuscript.Figure S1. Phylogenetic analysis of internal genes of Thai H4N6 and H4N9 viruses and other H4N6 and H4N9 influenza A viruses. The phylogenetic tree was generated using the neighbor-joining algorithm. Bootstrap analysis with 1000 replicates and posterior probability from BMCMC analysis were performed for confirming tree topology . The H4N6 and H4N9 influenza viruses characterized in the study are present in triangle.Click here for file


Therapies targeting MAPK and AKT/mTOR signaling are currently being evaluated in clinical trials for several tumor types. However, recent studies suggest that these therapies may be limited due to acquired cancer cell resistance and a small therapeutic index between normal and cancer cells. The identification of novel proteins that are involved in MAPK or AKT/mTOR signaling and differentially expressed between normal and cancer cells will provide mechanistically distinct therapeutic targets with the potential to inhibit these key cancer-associated pathways. We recently identified FAM83B as a novel, previously uncharacterized oncogene capable of hyperactivating MAPK and mTOR signaling and driving the tumorigenicity of immortalized human mammary epithelial cells (HMEC). We show here that elevated FAM83B expression also activates the PI3K/AKT signaling pathway and confers a decreased sensitivity to PI3K, AKT, and mTOR inhibitors. FAM83B co-precipitated with the p85α and p110α subunits of PI3K, as well as AKT, and increased p110α and AKT membrane localization, consistent with elevated PI3K/AKT signaling. In tumor-derived cells harboring elevated FAM83B expression, ablation of FAM83B decreased p110α and AKT membrane localization, suppressed AKT phosphorylation, and diminished proliferation, AIG, and tumorigenicity in vivo. We propose that the level of FAM83B expression may be an important factor to consider when combined therapies targeting MAPK and AKT/mTOR signaling are used. Moreover, the identification of FAM83B as a novel oncogene and its integral involvement in activating PI3K/AKT and MAPK provides a foundation for future therapies aimed at targeting FAM83B in order to suppress the growth of PI3K/AKT- and MAPK-driven cancers. The acquisition of persistent growth signaling is a hallmark of cellular transformation that can be conferred by an ever increasing number of mechanisms ;2. OftenIn numerous breast cancer subtypes, elevated RTK and PI3K signaling is an important driver of transformation . As withRecently, we identified FAM83B  in a forward genetic screen for genes that drive human mammary epithelial cell (HMEC) transformation . FurtherWe recently identified FAM83B in a Validation-Based Insertional Mutagenesis (VBIM) screen based on its ability to promote the anchorage-independent growth (AIG) and tumorigenicity of immortalized HMEC in a manner similar to RAS-G12V . SubsequGFP (G) or FAM83B (B) before assessing cell number. Western analysis confirmed the expression of CA-CRAF and CA-MEK1 in the RAS-G12V-transformed HME1 cells. As expected, ablation of FAM83B resulted in a significant reduction in cell number , PI3K (Y40C), or RAL-GEF (E37G). Western analysis confirmed the expression of the RAS-V12G proteins . Both FAin vivo . This phin vivo . Taken tIn addition to activating CRAF signaling, FAM83B expression results in elevated conventional phospholipase D (PLD) activity. Inhibition of either CRAF or PLD1 suppresses FAM83B-mediated transformation . Therefoet. al demonstrated that another member of this family, FAM83A, can activate the PI3K signaling axis, leading to AKT phosphorylation [FAM83B is one of eight members of a protein family (FAM83), based solely on the presence of an N-terminal Domain of Unknown Function (DUF1669). A recent report by Lee rylation . To deteHaving defined PI3K/AKT signaling as a key effector pathway that cooperates with MAPK signaling to transform HME1 cells, we next sought to confirm that FAM83B-mediated transformation requires sustained PI3K/AKT activity. HME1 cells expressing GFP or FAM83B were analyzed for AIG in the presence of a PI3K inhibitor (LY294002) or an AKT inhibitor (MK2206) Fig. . FAM83B-FAM83B (B) or GFP (G) was delivered to HCC1937 and MDA468 breast cancer cells (harboring elevated EGFR). The efficiency of FAM83B knock-down was examined by Western analysis  with lentiviruses expressing shRNAs targeting FAM83B (B) or GFP (G), injected NOD/SCID mice, and measured tumor formation after two weeks. Again, suppression of FAM83B resulted in a significant inhibition of both tumor volume and tumor weight when compared to control, shGFP-expressing cells , or constitutively active PI3K (CA-PI3K), when combined with constitutively active MEK1 (CA-MEK) could prevent the growth suppression observed following FAM83B ablation. For this, MDA468 cells expressing CA-AKT and CA-MEK or CA-PI3K and CA-MEK were created, infected with lentiviruses encoding FAM83B-shRNA, and AIG was measured Fig. . While sTo define the mechanism by which FAM83B activates PI3K/AKT signaling, we examined whether PI3K signaling components co-precipitated with FAM83B. We first examined whether FAM83B co-precipitated PI3K subunits by co-expressing FAM83B together with either the p85α regulatory subunit of PI3K or the p110α catalytic subunit. FAM83B was able to co-precipitate with both the p85α and p110α subunits Fig.  and 6B. Understanding the mechanism by which elevated FAM83B expression drives cellular transformation will provide the foundation for future therapies aimed at suppressing the key functions of this novel oncogene. Previously, we demonstrated that FAM83B expression elevates MAPK activity by increasing CRAF membrane localization. Based on our observations, we put forth a hypothesis that FAM83B was a key regulator of RAF/MAPK signaling, and that its function was simply to connect upstream EGFR/RAS with RAS/MAPK activation. Surprisingly, when we began examining the requirements for HME1 transformation, we noted that the expression of activated CRAF or MEK1 failed to induce AIG comparable to FAM83B. Additionally, the expression of RAS-G12V mutants only capable of activating a single downstream effector pathway  in HME1 cells failed to induce AIG comparable to RAS-G12V or FAM83B expressing HME1 cells. Therefore, FAM83B-mediated transformation could not be recapitulated by any single RAS effector.During our original characterization of FAM83B function, we focused on a phospholipase D-like motif in the N-terminal Domain of Unknown Function 1669 (DUF1669), as it was the only clear functional motif. Indeed, we observed FAM83B-expressing cells had elevated PLD activity; however, this was not due to FAM83B itself but the hyperactivation of PLD1 . Here, wFAM83B is a member of an 8 member protein family (FAM83) that shares the highly conserved DUF1669. In addition to our identification of FAM83B, Lee et al. identified FAM83A in a cDNA library screen for proteins that confer resistance to EGFR tyrosine kinase inhibitors. Their study identified an interaction between FAM83A and the PI3K regulatory subunit p85α to activate the PI3K signaling axis, leading to AKT phosphorylation . TherefoThe increased PI3K/AKT activity observed in FAM83B-expressing cells, together with the activation of MAPK signaling indicates that FAM83B is an important protein in RAS effector activation Fig. . AdditioThe subcellular protein fractionation kit was purchased from Thermo Scientific and the standard protocol was used. RAF Kinase Inhibitor I (GW5074), U0126, LY294002, and Rapamycin were purchased from Calbiochem, and MK2206 was purchased from Selleck Bio., and Matrigel was purchased from BD Biosciences.pBabe-puro-cRAF-22W was kindly provided by Channing J. Der . pcDNA5/TO-myc-PLD1 and pcDNA5/TO-myc-PLD2 were provided by H. Alex Brown  and wasRetroviruses were produced as described ;19. Brie2 at 37°C in Medium 171 with mammary epithelial growth supplement (Cascade Biologics) and 50 units/mL of penicillin and 50 μg/mL of streptomycin sulfate  as described [2 in DMEM  with 5% fetal bovine serum and 50 units/mL of penicillin and 50 μg/mL of streptomycin sulfate .hTERT-HME1 cells were purchased from (Clontech) and grown in a humidified atmosphere containing 5% COescribed . HCC19374. Cell extracts containing equal quantities of proteins, determined by the Bradford method, were separated by SDS-PAGE (8–12.5% acrylamide) and transferred to polyvinylidene difluoride membranes (Millipore). Antibodies to HA (F-7), MYC (9E10), HRAS(C-20), and PC-PLD1 (F-12) were from Santa Cruz Biotechnology, antibodies to ß-actin (pan Ab-5) were from Neomarkers, antibodies to glyceraldehyde-3-phosphate dehydrogenase were from Calbiochem, antibodies to FLAG (M2) were from Sigma-Aldrich and antibodies for ERK1/2, P-ERK1/2 (Thr202/Tyr204), AKT, P-AKT (Ser473), CRAF, STAT3, EGFR, PI3K, p85, and MEK1/2 were from Cell Signaling. Monoclonal FAM83B antibodies (clones 7D11 and 12G11) were generated against recombinant FAM83B protein . Primary antibodies were detected with goat anti-mouse or goat anti-rabbit conjugated to horseradish peroxidase (Hoffman-La Roche), using enhanced chemiluminescence (Perkin-Elmer).Immunoprecipitation experiments were carried out as previously described with exceptions as noted ;21. FAM85 cells were suspended in 0.6% type VII agarose (Sigma) and plated onto a bottom layer of 1.2% agar in a 60mm plate in triplicate as described [5 cells were plated per 60mm dish and medium was changed every 3 days until cells were analyzed after 3 weeks. The specific pharmacological PI3K inhibitor LY294002 was obtained from Sigma Aldrich and the AKT inhibitor MK2206 was obtained from Selleck Chemicals. Both compounds were added to the medium during feedings for indicated experiments.For hTERT-HME1 cells, 1 × 10escribed . The medhTERT-HME1, HCC1937, and MDA468 cells were plated in triplicate at 20,000 cells/well in triplicate and cell number was determined on a Beckman Coulter counter. The RAF inhibitor GW5074, mTOR inhibitor Rapamyicn, MEK1/2 inhibitor U0126, and PI3K inhibitor LY294002 were obtained from Sigma-Aldrich; the AKT inhibitor was obtained from Selleck Chemicals and added to the indicated experiments.5 cells are embedded in 880 μl lrBM and allowed to solidify for 1 hour. The solidified lrBM cultures are reefed with 1mL of complete growth media, with refeedings every 4 days for 12 days. Phase contrast images were taken at day 12 and total acini were quantified using an automated multi-panel scanning microscope, and the digital images were analyzed using MetaMorph image quantification software.Growth factor reduced lrBM was purchased from BD Biosciences. LrBM cultures were performed by overlaying 120 μl of growth factor reduced lrBM onto 6 well chamber dishes and allowing it to solidify for 30 minutes at 37ºC in a cell culture incubator. Approximately 1×106) cancer cells were suspended in a 1:1 mixture of media and matrigel (BD Biosciences) and injected subcutaneously into mice (6-8 weeks of age) that were sublethally irradiated with 300 rad 4 hours prior to injection. Tumor volume was calculated with the formula 4/3πr3 and student t-tests were performed.NOD/SCID mice were bred and maintained under defined conditions at the Athymic Animal and Xenograft Core facility at the Case Western Reserve University, Case Comprehensive Cancer Center an accredited facility that acts in compliance with NIH guidelines and provides veterinary care by several full time veterinary personnel. HCT116 (1×10


The complete genomic sequence of a new H9N2 avian influenza virus (AIV), isolated in northwestern China, was determined. Sequence and phylogenetic analyses based on the sequences of eight genomic segments revealed that the isolate is phylogenetically related to the Y280-like sublineage. During the last decade, H9N2 avian influenza viruses (AIVs) have circulated worldwide in poultry and caused economic losses in the poultry industry in many countries , 2. AsiaIn this study, the full-length sequences of eight genomic segments of a new H9N2 AIV isolate, A/chicken/Shaanxi/11/2012 (H9N2), were determined. Sequence and phylogenetic analyses indicated that the isolate is phylogenetically related to the Y280-like sublineage. In the present study, we found that the hemagglutinin (HA), neuraminidase (NA), and nonstructural (NS) genes of the new isolate share 92 to 98.0% nucleotide homologies with those of the H9N2 AIVs. More importantly, the nucleoprotein (NP) gene is closely related to that of the H5N1 AIVs isolated in eastern China. The matrix (M) gene has 92.0 to 93.1% nucleotide similarity with a H6N1 virus isolated from Guangzhou province in 2004, indicating that complicated reassortments of AIVs occurred in China.335RSSRGLF341 sequences at the cleavage site in the HA protein indicate that the isolate is a low-pathogenic type (LPAIV) (The (LPAIV) . Seven p (LPAIV) . Two ami (LPAIV) . H9N2 AI (LPAIV) . InteresThe new isolate does not contain the mutation E62K or D701N  in the RThe GenBank accession numbers of the new H9N2 AIV isolate from Shaanxi province are shown in


Understanding swine influenza virus (SIV) ecology has become more and more important from both the pig industry and public health points of views. However, the mechanism whereby SIV occurs in pig farms is not well understood. The purpose of this study was to develop a proper strategy for SIV surveillance.We conducted longitudinal monitoring in 6 farrow-to-finish farms in the central region of Thailand from 2008 to 2009. Nasal swabs and serum samples were collected periodically from clinically healthy pigs consisting of sows, fattening pigs, weaned piglets and pigs transferred from other farms. A total of 731 nasal swabs were subjected to virus isolation and 641 serum samples were subjected to detection of SIV antibodies against H1 and H3 subtypes using the hemagglutination inhibition test and ELISA. Twelve SIVs were isolated in this study and eleven were from piglets aged 4 and 8 weeks. Phylogenetical analysis revealed that SIVs isolated from different farms shared a common ancestor. Antibodies against SIVs were detected in fattening pigs on farms with no SIV isolation in the respective periods studied. These observations suggested that piglets aged 8 weeks or younger could be a main target for SIV isolation. Farm-to-farm transmission was suggested for farms where pigs from other farms are introduced periodically. In addition, antibodies against SIVs detected in fattening pigs could be a marker for SIV infection in a farm.The present study provided important information on SIV surveillance that will enable better understanding of SIV ecology in farrow-to-finish farms. Swine influenza virus (SIV) is one of the pathogens that cause respiratory diseases accompanied with coughing and sneezing in pigs . This viA novel H1N1 virus, later designated as a pandemic (H1N1) 2009 (H1N1pdm) virus, was first identified in April 2009 when it caused the first influenza pandemic in humans in the 21st century . Origin Ecology of SIVs is highly complicated due to multiple genetic reassortments, although three subtypes H1N1, H1N2 and H3N2 are dominant in swine populations . Triple The pig industry in Thailand has been expanding rapidly as one of the major livestock industries since the 1970s . Our preForty nasal swabs were collected from 20 sows aged from 1 to 2 years, 10 fattening pigs aged 12 weeks and 10 weaned piglets aged 9 weeks in Farm A in January 2008. Five farms, B, C, D, E and F, were visited periodically to collect nasal swabs and blood samples three or more times from June 2008 to November 2009 , Thailand as described previously . BrieflyDirect sequencing of the PCR products and phylogenetic analysis of the viruses isolated in this study were carried out as described previously .Vibrio Cholerae  to remove any non-specific inhibitors of hemagglutination, and were then inactivated at 56°C for 30 min. Next, the serum samples were absorbed with packed chicken red blood cells for 60 min at room temperature. A cut off value of 1:40 was adopted to avoid false-positive cases due to non-specific reactions in the HI tests. Commercial ELISA kits  were used to detect antibodies against 'classical' H1 and 'human-like' H3 SIV HAs according to the manufacturer's instructions.Serum was obtained from the collected blood samples by centrifugation for 10 min at 3500 rpm. All of the serum samples were subjected to the hemagglutination inhibition (HI) test and ELISA. Antigens used for the HI tests were A/swine/Ratchaburi/NIAH550/2003 , A/swine/Ratchaburi/NIAH101942/2008 , A/swine/Saraburi/NIAH107725-28/2008 , and A/swine/Chachoengsao/2003  [AB620160- AB620211.The sequences determined in this study are available from GenBank under accession numbers A total of 731 nasal swabs and 641 serum samples were collected from six farms in the Ratchaburi, Saraburi and Singburi provinces in the central region of Thailand  (Sara107725-28) and A/swine/Saraburi/NIAH109713-36/2009 (H3N2) (Sara109713-36), were isolated from Farm B. Sara107725-28 was isolated from a weaned piglet at the age of 4 weeks on June 9, 2008. Sara109713-36 was isolated from an 8-week-old introduced piglet on July 1, 2009. They shared high nucleotide homologies of more than 96.7% in each segment and their gene constellations were similar and identical with that of A/swine/Ratchaburi/NIAH874/05 (H3N2), reported elsewhere   in a farm. Antibodies against those pathogens were detected at high rates in the sow population in a farm [The presence of seropositive fattening pigs in Farms C and E suggested SIV infection, however, no SIV was isolated. Sero-positive reaction was always observed against H1 SIVs in fattening pigs in Farm C, and an apparent increase in the number of fattening pigs seropositive against H1 SIVs was observed in January 2009 in Farm E. The reason why SIVs were not isolated in these farms may be explained by the fact that SIVs could circulate continuously in those farms with a prevalence rate lower than the detection limit rate in our sampling numbers. Sows were suggested to be a reservoir for continuous circulation of some respiratory pathogens ; Sara100761-22: A/swine Saraburi/NIAH100761-22/2009 (H1N1); Sara116625-11: A/swine/Saraburi/NIAH116625-11/2009 (H1N1); Sara116627-24: A/swine/Saraburi/NIAH116627-24/2009 (H1N1); Sara107725-28: A/swine/Saraburi/NIAH107725-28/2008 (H3N2); Sara109713-36: A/swine/Saraburi/NIAH109713-36/2009 (H3N2)SIV: Swine influenza virus; SAα2,6Gal: Sialic acid linked to galactose by α2,6 linkage; SAα2,3Gal: Sialic acid linked to galactose by α2,3 linkage; H1N1pdm: pandemic (H1N1) 2009; HI test: hemagglutination inhibition test; TCIDThe authors declare that they have no competing interests.NT, YH, TH and YU carried out virus isolation from the specimens. NT carried out genetical and serological analyses. SP and RR coordinated the pig farm visits. All authors participated in the sampling specimens at the farms. NT and TS designed the study and draft the manuscript. All authors read and approved the final manuscript.Supplementary figure S1; Supplementary figure S2; Supplementary figure S3; Supplementary figure S4Click here for file