Evaluation of Automated Magnetic Bead-Based DNA Extraction for Detection of Short Tandem Repeat Expansions With Nanopore Sequencing.

BACKGROUND: Long-read technologies such as nanopore sequencing provide new opportunities to detect short tandem repeat expansions. Therefore, a DNA extraction method is necessary that minimizes DNA fragmentation and hence allows the identification of large repeat expansions. In this study, an automated magnetic bead-based DNA extraction method and the required EDTA blood storage conditions as well as DNA and sequencing quality were evaluated for their suitability for repeat expansion detection with nanopore sequencing. METHODS: DNA was extracted from EDTA blood, and subsequently, its concentration, purity, and integrity were assessed. DNA was then subjected to nanopore sequencing, and quality metrics of the obtained sequencing data were evaluated. RESULTS: DNA extracted from fresh EDTA blood as well as from cooled or frozen EDTA blood revealed high DNA integrity whereas storage at room temperature over 7 days had detrimental effects. After nanopore sequencing, the read length N50 values of approximately 9 kb were obtained, and based on adaptive sampling of samples with a known repeat expansion, repeat expansions up to 10 kb could be detected. CONCLUSION: The automated magnetic bead-based DNA extraction was sufficient to detect short tandem repeat expansions, omitting the need for high-molecular-weight DNA extraction methods. Therefore, DNA should be extracted either from fresh blood or from blood stored in cooled or frozen conditions. Consequently, this study may help other laboratories to evaluate their DNA extraction method regarding the suitability for detecting repeat expansions with nanopore sequencing.

Carnosine inhibits glioblastoma growth independent from PI3K/Akt/mTOR signaling.

Glioblastoma is a high-grade glioma with poor prognosis even after surgery and standard therapy. Here, we asked whether carnosine (ß-alanyl-L-histidine), a naturally occurring dipeptide, exert its anti-neoplastic effect on glioblastoma cells via PI3K/Akt/mTOR signaling. Therefore, glioblastoma cells from the lines U87 and T98G were exposed to carnosine, to the mTOR inhibitor rapamycin and to the PI3K inhibitor Ly-294,002. Pyruvate dehydrogenase kinase (PDK4) expression, known to be a target of PI3K/Akt/mTOR, and which is also affected by carnosine, was analyzed by RT-qPCR, and reporter gene assays with the human PDK4 promoter were performed. Cell viability was assessed by cell-based assays and mTOR and Akt phosphorylation by Western blotting. Rapamycin and Ly-294,002 increased PDK4 mRNA expression in both cell lines but significance was only reached in U87. Carnosine significantly increased expression in both lines. A significant combinatorial effect of carnosine was only detected in U87 when the dipeptide was combined with Ly-294,002. Reporter gene assays revealed no specific effect of carnosine on the human PDK4 promoter, whereas both inhibitors increased reporter gene expression. Rapamycin reduced phosphorylation of mTOR, and Ly-294,002 that of Akt. A significant reduction of Akt phosphorylation was observed in the presence of carnosine in U87 but not in T98G, and carnosine had no effect on mTOR phosphorylation. Cell viability as determined by ATP in cell lysates was reduced only in the presence of carnosine. We conclude that carnosine's anti-neoplastic effect is independent from PI3K/Akt/mTOR signaling. As the dipeptide reduced viability in tumor cells that do not respond to PI3K or mTOR inhibitors, it appears to be worth to further investigate the mechanisms by which carnosine exerts its anti-tumor effect and to consider it for therapy, especially as it is a naturally occurring compound that has already been used for the treatment of other diseases without indication of side-effects.