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Background: Cancer stem cells (CSCs), a subpopulation in tumors, are known to cause drug resistance, tumor recurrence and metastasis. Based on the characteristic formation of mammospheres in in vitro conditions, the mammosphere formation assay has become an essential tool for quantifying CSC activity in breast cancer research. However, manual counting of mammospheres is a time-consuming process that is not amenable to high-throughput screening, and there are occasional inaccuracies in the process of determining the mammosphere diameter. In this study, we proposed a novel automated counting method of mammosphere using the National Institute of Standards and Technology (NIST)'s Integrated Colony Enumerator (NICE) with a screening of protein kinase library. Methods: Human breast cancer cell line MCF-7 was used for evaluation of tumor sphere efficiency, migration, and phenotype transition. Cell viability was assessed using MTT assay, and CSCs were identified by an analysis of CD44 expression and ALDEFLUOR assay using flow cytometry. Automated counting of mammosphere using NICE program was performed with a comparison to the result of manual counting. After identification of inhibitors to ameliorate CSC formation by screening a library of 79 protein kinase inhibitors using automated counting in primary, secondary and tertiary mammosphere assay, the effect of selected kinase inhibitors on migration, colony formation and epithelial- to-mesenchymal transition (EMT) of MCF-7 cells was investigated. Results: Automated counting of mammosphere using NICE program was an easy and less time-consuming process (<1 min for reading 6-well plate) which provided a comparable result with manual counting. Inhibition of calcium/ calmodulin-dependent protein kinase II (CaMKII), Janus kinase-3 (JAK-3), and IкB kinase (IKK) were identified to decrease the formation of MCF-7-derived CSCs in primary, secondary and tertiary mammosphere assay. These protein kinase inhibitors alleviated TGF-β1-induced migration, colony formation and EMT of MCF-7 cells. Conclusions: We have developed a novel automated cell-based screening method which provided an easy, accurate and reproducible way for mammosphere quantification. This study is the first to show the efficacy of an automated medium-throughput mammosphere-counting method in CSC-related research with an identification of protein kinase inhibitors to ameliorate CSC formation. [ABSTRACT FROM AUTHOR]

Background: Bovine Leukemia virus (BLV) infection of cattle has been reported in Korea for more than three decades. However, to date, there have been few studies regarding Korean BLV since 1980s. Thus, the purpose of this study is to perform a diagnosis and molecular characterization of BLV strains circulating in Korea and to estimate genetic diversity of different genotypes of BLV. Method: To investigate the distribution of BLV variants in the world and assess the evolutionary history of Korean BLV isolates, a comprehensive molecular analysis of the BLV env gp51 gene was conducted using recent worldwide BLV isolates. The isolates included 50 samples obtained from two cattle farms in southeastern Korea in 2014. Results: Sequence and phylogenetic analyses of partial 444-nt fragment sequences and complete gp51 sequences of BLV revealed eight distinct genotypes of BLV showing geographic distribution of the world. Most Korean BLV isolates were found to belong to genotype 1 which is a major genotype prevailed throughout the world, and only four isolates from one farm were classified as genotype 3 related to the US and Japan isolates. Analysis of amino acids of Korean BLV isolates showed several sequence substitutions in the leader peptide, conformational epitope, and neutralizing domain regions. The observations suggest the possibility of affecting on viral infectivity and formation. Conclusion: Korean BLV isolates showed the close relationship to genotype 1 and 3. Further study to identify the diversity of BLV circulating in Korea is necessary with samples collected nationwide because this study is the first report of BLV genotype 3 being in circulation in Korea. [ABSTRACT FROM AUTHOR]