The significant differences in DNA methylation that are considered to be a biomarker for the diagnosis of cancer are a barrier to the application of biomarkers in the clinical field. In the present study, new primers were designed and further standard controls were set up to validate the accuracy of the methylation‑specific PCR (MSP), a method widely used to analyze DNA methylation. By analyzing the methylation pattern of breast cancer 1 (BRCA1) and estrogen receptor (ER) in 60 patients with breast cancer, the number of cases of methylated BRCA1 and ER detected by the primer was 7/60 and 21/60, respectively, whereas that detected by the previous widely used primers was 25/60 and 47/60, respectively. Sequencing of the MSP products indicated that the 18 and 26 false-positive methylations of BRCA1 and ER, respectively, were due to insufficient validation of the previously used primers. Thus, the present study proposes that all studies based on the MSP approach should incorporate more controls to validate the precision of the MSP primers.

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The MSP-DB dispels the weakness of MSP and increases the sensitivity to simultaneous methylation analysis of multiple genes. The MSP-DB is advantageous for the promotion of DNA methylation markers in progressing quickly toward clinical application.

Objectives: To examine the safety of a BaciMix product, containing two strains of Bacillus subtilis BS 304.04 and Bacillus coagulans BC 304.06 on mice and rats. Materials and methods: The acute toxicity using the Litchfield-Wilcoxon method on Swiss mice and semi-chronic toxicity of the BaciMix product on Wistar rats was performed. Results: LD50 of the mice which were administered orally with the highest dose of 4 x 1011 CFU/kg of the product was not determined. The BaciMix product was well-tolerated and did not show any effects on the growth or food intake in animals. The differences in the number of red blood cells, white blood cells, hemoglobins, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, cholesterol, protein, bilirubin and creatinine were not statistically significant in rats serum dosed continuously for 28 days with of 1.68 x 109 CFU/kg and 8.4 x 109 CFU/kg of the product. In addition, no histopathological abnormalities or changes were observed in all the groups of animals. Conclusion: These results suggested that the BaciMix product could be safe for human use.

Chito-oligosaccharide (COS), one of remakable derivatives of chitosan, has almost chitosan’s biological function but better activities in antibacteria, antifungi and plant elicitor… Producing COS by biological enzymes shows many advantages in comparision with physical and chemical methods. In our study, COS was prepared using chitin/chitosan-hydrolyzing enzyme from actinomycetes. By analysis of 16S rDNA sequence, the enzyme producer VTCC 940003 belonged to Streptomyces macrosporeus. The strain’s ability to synthesize the enzyme having chitin/chitosan-hydrolyzing activity was first detected by agar plate method and then its activity equivalent to chitinase activity was determined using dinitrosalicylic acid (DNS). The enzyme activity in the strain culture was 24 ± 0.05 U/ml. The COS was prepared by using the enzyme from S. macrosporeus VTCC 940003 to hydrolyze chitosan. The degree of polymerization (DP) of the obtained COS was mainly DP3-DP5. At the concentration of 0,3‰ the COS inhibited 98% of spore germination of Fusarium oxysporum after 24 hours and this activity remained after 48 hours.