Alteration in stemness causes exclusivity between Epstein-Barr virus-positivity and microsatellite instability status in gastric cancer.

BACKGROUND: Gastric cancer (GC) is a leading cause of cancer morbidity and mortality worldwide. This is due to the heterogeneous features of GC, which consist of a diverse molecular phenotype. Epstein-Barr virus (EBV)-positive GC and microsatellite instability (MSI)-high GC encompass similar epigenetic traits, including high levels of DNA methylation in CpG islands; however, EBV-positive and MSI-high GCs are mutually exclusive. We aimed to elucidate the underlying mechanism of this exclusivity. METHODS: We knocked out MLH1 in EBV-positive GC cell lines SNU-719 and NCC24 via CRISPR-Cas9, and evaluated the modified cellular properties in vitro and in vivo. The MSI status of each cell line was screened with two marker capillary electrophoresis, and further diagnosed with five marker capillary electrophoresis and parallel sequencing using 21 markers. RESULTS: Initial evaluation showed that cell growth, migration, invasion, and MSI status were not affected by MLH1 silencing. However, with prolonged passage, GC cell lines gradually gained MSI and NCC24 cells were transformed to EBV-positive/MSI-high GC cells after 12 months. Furthermore, MLH1 silencing reduced tumor stemness in SNU-719 and NCC24 regardless of the MSI status in vitro and in vivo. CONCLUSIONS: Our findings suggest that EBV-positivity and MSI-high status are mutually exclusive due to the immediate disadvantage in tumor stemness when MLH1 is silenced, whereas the establishment of MSI-high status in EBV-positive GCs required a longer period.

Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene.

Successful PCR starts with proper priming between an oligonucleotide primer and the template DNA. However, the inevitable risk of mismatched priming cannot be avoided in the currently used primer system, even though considerable time and effort are devoted to primer design and optimization of reaction conditions. Here, we report a novel dual priming oligonucleotide (DPO) which contains two separate priming regions joined by a polydeoxyinosine linker. The linker assumes a bubble-like structure which itself is not involved in priming, but rather delineates the boundary between the two parts of the primer. This structure results in two primer segments with distinct annealing properties: a longer 5'-segment that initiates stable priming, and a short 3'-segment that determines target-specific extension. This DPO-based system is a fundamental tool for blocking extension of non-specifically primed templates, and thereby generates consistently high PCR specificity even under less than optimal PCR conditions. The strength and utility of the DPO system are demonstrated here using multiplex PCR and SNP genotyping PCR.

Heritability of muscle mass in Korean parent-offspring pairs in the Fifth Korean National Health and Nutrition Examination Survey (KNHANES V).

OBJECTIVES: Decreased muscle mass is known to be associated with several serious medical conditions. We analyzed the Fifth Korean National Health and Nutrition Examination Survey (KNHANES V, 2010-2011) to estimate the heritability of muscle mass in Korean parent-offspring pairs. STUDY DESIGN: Cross-sectional. MAIN OUTCOME MEASURES: A total of 1233 parents (average age 57.67 ±â€¯8.50 years) and 917 offspring (average age 29.10 ±â€¯7.57 years) from 743 families were included in the analysis. Muscle mass was estimated based on three different indices: appendicular skeletal muscle mass (ASM) measured with a dual-energy X-ray absorptiometry (DXA), weight-adjusted ASM (SMI), and height-adjusted ASM (RASM). The heritability was estimated by employing the maximum-likelihood variance components implemented in Sequential Oligogenic Linkage Analysis Routines (SOLAR). The best-fitting model was determined out of four polygenic models. Pearson's partial correlation coefficient was also calculated using the muscle mass indices to further study the association between father or mother and son or daughter pairs. RESULTS: The heritability estimates of the muscle mass indices ranged from 55% to 80% (all p < 0.01). The correlation coefficient of father and offspring ranged from 0.11 to 0.40, while that of mother and offspring ranged from 0.23 to 0.43 (all p < 0.01). CONCLUSIONS: The heritability estimates of muscle mass in Koreans are large and significant, suggesting that parental muscle mass is an important predictor of the offspring's muscle mass. The result implies that there may be a genetic factor partly determining muscle mass.

Annealing control primer system for identification of differentially expressed genes on agarose gels.

We developed GeneFishing technology, an improved method for the identification of differentially expressed genes (DEGs) using our novel annealing control primer (ACP) system. Because of high annealing specificity during PCR using the ACP system, the application of the ACP to DEG discovery generates reproducible, authentic, and long (100 bp to 2 kb) PCR products that are detectable on agarose gels. To demonstrate this method for gene expression profiling, Gene-Fishing technology was used to detect genes that are differentially expressed during development using total RNAs isolated from mouse conceptus tissues at 4.5-18.5 days of gestation. Ten DEGs (DEG1-10) were isolated and confirmed by Northern blot hybridization. The sequence analysis of these DEGs showed that DEG6 and DEG10 are unknown genes.

Annealing control primer system for improving specificity of PCR amplification.

A novel primer designed to improve the specificity of PCR amplification, called the annealing control primer (ACP), comprises a tripartite structure with a polydeoxyinosine [poly(dI)] linker between the 3' end target core sequence and the 5' end nontarget universal sequence. We show that this ACP linker prevents annealing of the 5' end nontarget sequence to the template and facilitates primer hybridization at the 3' end to the target sequence at specific temperatures, resulting in a dramatic improvement of annealing specificity. The effect of this linker is demonstrated by the incorporation of ACP sequences as primers during the amplification of target nucleotide sequence and as hybridization probes in the genotyping of single nucleotide polymorphisms. This is the first report to show that a poly(dI) linker between two different sequences of ACP forms a bubble-like structure and disrupts or destabilizes DNA duplex formation at certain annealing temperatures.

Prevalence of human papillomavirus infection and genotype distribution determined by the cyclic-catcher melting temperature analysis in Korean medical checkup population.

Although cytology screening has reduced the incidence and mortality rate of cervical cancer significantly, its usefulness is limited to samples from the site of the lesion, resulting in its low sensitivity and unsuitability for use in medical checkups. The purpose of the present study was to evaluate the prevalence of HPV infection using genotype distribution and to analyze the correlation of the HPV DNA test results with cytological results. We also evaluated the benefits of quantitative information obtained from cyclic-catcher melting temperature analysis (CMTA) in screening for cervical cancer. We performed cyclic-CMTA using Anyplex™ II HPV28 Detection in combination with cervical cytology for 2,181 subjects. The following HPV positivity types were detected using cyclic-CMTA and HPV positivity was found to increase together with the severity of the cytology results: (1) For 419 HPV positive specimens, HPV DNA was detected in 18.1% of normal specimens, 78.3% of ASCUS, and all of LSIL and HSIL; (2) high-risk HPV DNAs were detected in 63.3% of normal (N=547), 65.9% of ASCUS (N=41), 76.9% of LSIL (N=13), and 88.9% of HSIL (N=9) among total detected HPV DNA regardless multiple detection; (3) multiple HPV genotypes were detected in 4.8% of normal specimens (N=2,146), 52.2% of ASCUS (N=23), 57.1% of LSIL (N=7), and 40.0% of HSIL (N=5). In addition, a high level of viral DNA was observed using cyclic-CMTA in all specimens beyond the LSIL stage according to cytology, while only 6% of specimens with normal cytology showed a correlation with viral quantitation by cyclic-CMTA. The combination of HPV genotyping with a quantitative assay and cytology will allow for a more accurate diagnosis of cervical cancer.