Spectrophotometric Detection of the BRCA1 Gene via Exponential Isothermal Amplification and Hybridization Chain Reaction of Surface-Bound Probes.

In this work, we demonstrated an ultrasensitive approach with a dual-amplification strategy for DNA assay based on isothermal exponential amplification (EXPAR) and the hybridization chain reaction (HCR). In the presence of target DNA, the hairpin probe DNA (HP1) recognized and partially hybridized with the target DNA to form double-stranded structures containing the full recognition sequences for nicking endonuclease and then initiated EXPAR. Under the reaction of EXPAR, a large number of single-stranded DNA (ssDNA) was produced in the circle of nicking, polymerization, and strand displacement. The resulting ssDNA can bind to the surface-bound probe on the well of the microplate and trigger the hybridization chain reaction, resulting in the production of numerous double-stranded DNA concatamers with biotin labeling. In the presence of streptavidin-conjugated horseradish peroxidase (HRP), the amplified signal can be detected by a spectrophotometer via HRP-catalyzed substrate 3,3'5,5'-tetramethylbenzidine (TMB). This proposed dual-amplification method provides a detection limit of 74.48 aM, which also exhibits good linearity ranging from 0.1 fM to 100 pM.

Structural and functional analysis of three ß-glucosidases from bacterium Clostridium cellulovorans, fungus Trichoderma reesei and termite Neotermes koshunensis.

ß-glucosidases (EC 3.2.1.21) cleave ß-glucosidic linkages in disaccharide or glucose-substituted molecules and play important roles in fundamental biological processes. ß-Glucosidases have been widely used in agricultural, biotechnological, industrial and medical applications. In this study, a high yield expression (70-250 mg/l) in Escherichia coli of the three functional ß-glucosidase genes was obtained from the bacterium Clostridium cellulovorans (CcBglA), the fungus Trichoderma reesei (TrBgl2), and the termite Neotermes koshunensis (NkBgl) with the crystal structures of CcBglA, TrBgl2 and NkBgl, determined at 1.9Å, 1.63Å and 1.34Å resolution, respectively. The overall structures of these enzymes are similar to those belonging to the ß-retaining glycosyl hydrolase family 1, which have a classical (α/ß)(8)-TIM barrel fold. Each contains a slot-like active site cleft and a more variable outer opening, related to its function in processing different lengths of ß-1,4-linked glucose derivatives. The two essential glutamate residues for hydrolysis are spatially conserved in the active site. In both TrBgl2 and NkBgl structures, a Tris molecule was found to bind at the active site, explaining the slight inhibition of hydrolase activity observed in Tris buffer. Manganese ions at 10mM exerted an approximate 2-fold enzyme activity enhancement of all three ß-glucosidases, with CcBglA catalyzing the most efficiently in hydrolysis reaction and tolerating Tris as well as some metal inhibition. In summary, our results for the structural and functional properties of these three ß-glucosidases from various biological sources open important avenues of exploration for further practical applications.

MiRNA-10b sponge: An anti-breast cancer study in vitro.

Breast cancer is a malignant tumor with the highest incidence among women. Breast cancer metastasis is the major cause of treatment failure and mortality among such patients. MicroRNAs (miRNAs) are a class of small molecular non-coding regulatory RNAs, which act as oncogenes or tumor suppressors in breast cancer. miRNA-10b has been found to exhibit a high expression level in advanced and metastatic breast cancer, and is closely related to breast cancer metastasis. An miRNA sponge is an mRNA with several repeated sequences of complete or incomplete complementarity to the natural miRNA in its 3' non-translating region. It acts as a sponge adsorbing miRNAs and ensures their separation from their targets and inhibits their function. The present study designed a sponge plasmid against miRNA-10b and transiently transfected it into high and low metastatic human breast cancer cell lines MDA-MB-231 and MCF-7, and analyzed the effects of the miRNA-10b sponge on the growth and proliferation, migration and invasion in these cell lines. qRT-PCR results found that the sponge plasmid effectively inhibited the expression of miRNA-10b, and upregulated the expression of the miRNA­10b target protein HOXD-10. The results from the CCK-8 assay found that the miRNA-10b sponge inhibited the growth of breast cancer cell lines MDA-MB-231 and MCF-7. Results of the plate cloning experiments indicated that the miRNA-10b sponge suppressed the colony formation of the MDA-MB-231 and MCF-7 cells. The results of wound healing and Transwell assays showed that the miRNA-10b sponge inhibited the migration and invasion of the breast cancer cell lines MDA-MB-231 and MCF-7. Our results demonstrated that the miRNA-10b sponge effectively inhibited the growth and proliferation of breast cancer MDA-MB-231 and MCF-7 cells. In addition, it also restrained the migration and invasion of human highly metastatic breast cancer MDA-MB-231 cells.

[Expression and clinical significance of nm23-H1, Fas and FasL in colorectal carcinoma tissues].

OBJECTIVE: To investigate the expression and clinical significance of nm23-H1, Fas and FasL in colorectal carcinoma tissues. METHODS: The expression levels of nm23-H1, Fas and FasL in 48 cases of colorectal carcinoma tissues and 15 cases of normal colorectal tissues were determined by SP immunohistochemical staining. RESULTS: Positive expression rates of nm23-H1, Fas and FasL in colorectal carcinoma were (25.0 +/- 2.6)%, (41.7 +/- 2.7) and (77.1 +/- 2.7)% respectively; the positive expression rates of nm23-H1 and Fas were lower than that in the normal colorectal tissues (P<0.01); the FasL expression rate in colorectal carcinoma tissues was higher than that in the normal colorectal tissues (P<0.01); the lower expression of nm23-H1 and Fas, and the higher expression of FasL in colorectal carcinoma were correlated with the type of carcinoma histology, lymph node metastasis, Dukes' stage and prognosis (P<0.01). CONCLUSION: These results suggest that the lower expression of nm23-H1 and Fas and higher expression of FasL may be used as a good indicator in evaluating lymph node metastasis and prognosis.