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BACKGROUND Accumulating evidence suggests that some long noncoding RNAs (lncRNAs) are involved in certain diseases,such as cancer. The lncRNA,CCDC26,is related to childhood acute myeloid leukemia (AML) because its copy number is altered in AML patients. RESULTS We found that CCDC26 transcripts were abundant in the nuclear fraction of K562 human myeloid leukemia cells. To examine the function of CCDC26,gene knockdown (KD) was performed using short hairpin RNAs (shRNAs),and four KD clones,in which CCDC26 expression was suppressed to 1% of its normal level,were isolated. This down-regulation included suppression of CCDC26 intron-containing transcripts (the CCDC26 precursor mRNA),indicating that transcriptional gene suppression (TGS),not post-transcriptional suppression,was occurring. The shRNA targeting one of the two CCDC26 splice variants also suppressed the other splice variant,which is further evidence for TGS. Growth rates of KD clones were reduced compared with non-KD control cells in media containing normal or high serum concentrations. In contrast,enhanced growth rates in media containing much lower serum concentrations and increased survival periods after serum withdrawal were observed for KD clones. DNA microarray and quantitative polymerase chain reaction screening for differentially expressed genes between KD clones and non-KD control cells revealed significant up-regulation of the tyrosine kinase receptor,KIT,hyperactive mutations of which are often found in AML. Treatment of KD clones with ISCK03,a KIT-specific inhibitor,eliminated the increased survival of KD clones in the absence of serum. CONCLUSIONS We suggest that CCDC26 controls growth of myeloid leukemia cells through regulation of KIT expression. A KIT inhibitor might be an effective treatment against the forms of AML in which CCDC26 is altered. View Publication

Paramyxovirus entry into cells requires fusion of the viral and cell membranes mediated by one of the major virus glycoproteins,the fusion (F) glycoprotein which transits from a metastable pre-fusion conformation to a highly stable post-fusion structure during the membrane fusion process. F protein refolding involves large conformational changes of the protein trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) from each protomer into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of the Pneumovirinae F proteins,and as extension of previous work (Palomo et al.,2014),a general strategy was designed to obtain polyclonal and particularly monoclonal antibodies specific of the 6HB motif of the Pneumovirinae fusion protein. The antibodies reported here should assist in the characterization of the structural changes that the F protein of human metapneumovirus or respiratory syncytial virus experiences during the process of membrane fusion. View Publication

Although embryonal proteins have been used as tumor marker,most are not useful for detection of early malignancy. In the present study,we developed mouse monoclonal antibodies against fetal lung of miniature swine,and screened them to find an embryonal protein that is produced at the early stage of malignancy,focusing on lung adenocarcinoma. We found an antibody clone that specifically stained stroma of lung adenocarcinoma. LC-MS/MS identified the protein recognized by this clone as dimethylarginine dimethylaminohydrolase 2 (DDAH2),an enzyme known for antiatherosclerotic activity. DDAH2 was found to be expressed in fibroblasts of stroma of malignancies,with higher expression in minimally invasive adenocarcinoma (MIA) and invasive adenocarcinoma than in adenocarcinoma in situ (AIS). Moreover,tumors with high stromal expression of DDAH2 had a poorer prognosis than those without. In vitro analysis showed that DDAH2 increases expression of endothelial nitric oxide synthase (eNOS),inducing proliferation and capillary-like tube formation of vascular endothelial cells. In resected human tissues,eNOS also showed higher expression in invasive adenocarcinoma than in AIS and normal lung,similarly to DDAH2. Our data indicate that expression of DDAH2 is associated with invasiveness of lung adenocarcinoma via tumor angiogenesis. DDAH2 expression might be a prognostic factor in lung adenocarcinoma. View Publication

Heartland virus (HRTV),a phlebovirus first isolated from two Missouri farmers in 2009,has been proposed to be transmitted to humans by the bite of infected Amblyomma americanum ticks. It is closely related to severe fever with thrombocytopenia syndrome virus (SFTSV) from China,another previously unrecognized phlebovirus that has subsequently been associated with hundreds of cases of severe disease in humans. To expand diagnostic capacity to detect HRTV infections,20 hybridoma clones secreting anti-HRTV murine monoclonal antibodies (MAbs) were developed using splenocytes from HRTV-inoculated AG129 alpha/beta and gamma interferon receptor-deficient mice. Nine of these MAbs were characterized herein for inclusion in future HRTV diagnostic assay development. All of the MAbs developed were found to be non-neutralizing and reactive to linear epitopes on HRTV nucleocapsid protein. MAb 2AF11 was found to be cross-reactive with SFTSV. View Publication

The efficient generation of hepatocytes from human pluripotent stem cells (hPSCs) requires the induction of a proper endoderm population,broadly characterized by the expression of the cell surface marker CXCR4. Strategies to identify and isolate endoderm subpopulations predisposed to the liver fate do not exist. In this study,we generated mouse monoclonal antibodies against human embryonic stem cell-derived definitive endoderm with the goal of identifying cell surface markers that can be used to track the development of this germ layer and its specification to a hepatic fate. Through this approach,we identified two endoderm-specific antibodies,HDE1 and HDE2,which stain different stages of endoderm development and distinct derivative cell types. HDE1 marks a definitive endoderm population with high hepatic potential,whereas staining of HDE2 tracks with developing hepatocyte progenitors and hepatocytes. When used in combination,the staining patterns of these antibodies enable one to optimize endoderm induction and hepatic specification from any hPSC line. View Publication

Protein kinase CK2 (formerly casein kinase II) is a serine/threonine kinase overexpressed in many human tumors,transformed cell lines,and rapidly proliferating tissues. Recent data have shown that many cancers involve inappropriate reactivation of Wnt signaling through ectopic expression of Wnts themselves,as has been seen in a number of human breast cancers,or through mutation of intermediates in the Wnt pathway,such as adenomatous polyposis coli or beta-catenin,as described in colon and other cancers. Wnts are secreted factors that are important in embryonic development,but overexpression of certain Wnts,such as Wnt-1,leads to proliferation and transformation of cells. We report that upon stable transfection of Wnt-1 into the mouse mammary epithelial cell line C57MG,morphological changes and increased proliferation are accompanied by increased levels of CK2,as well as of beta-catenin. CK2 and beta-catenin co-precipitate with the Dvl proteins,which are Wnt signaling intermediates. A major phosphoprotein of the size of beta-catenin appears in in vitro kinase reactions performed on the Dvl immunoprecipitates. In vitro translated beta-catenin,Dvl-2,and Dvl-3 are phosphorylated by CK2. The selective CK2 inhibitor apigenin blocks proliferation of Wnt-1-transfected cells,abrogates phosphorylation of beta-catenin,and reduces beta-catenin and Dvl protein levels. These results demonstrate that endogenous CK2 is a positive regulator of Wnt signaling and growth of mammary epithelial cells. View Publication