No abstract

A theoretical analysis is presented with experimental confirmation to conclusively demonstrate the critical role that annealing plays in efficient PCR amplification of GC-rich templates. The analysis is focused on the annealing of primers at alternative binding sites (competitive annealing) and the main result is a quantitative expression of the efficiency (η) of annealing as a function of temperature (TA), annealing period tA) and template composition. The optimal efficiency lies in a narrow region of TA and tA for GC-rich templates and a much broader region for normal GC templates. To confirm the theoretical findings, the following genes have been PCR amplified from human cDNA template: ARX, and HBB (with 78.72% and 52.99% GC respectively). Theoretical results are in excellent agreement with the experimental findings. Optimum annealing times for GC-rich genes lie in the range of 3 to 6 seconds and depend on annealing temperature. Annealing times greater than 10 seconds yield smeared PCR amplified products. The non-GC-rich gene did not exhibit this sensitivity to annealing times. Theory and experimental results show that, shorter annealing times are not only sufficient but also necessary for efficient PCR amplification of GC-rich templates.

This study provides evidence that low-intensity ultrasound directly affects nuclear processes, and the magnitude of the effect varies with frequency. In particular, we show that the transcriptional induction of first load-inducible genes, which is independent of new protein synthesis, is frequency dependent. Bovine chondrocytes were exposed to low-intensity below the cavitational threshold) ultrasound at 2,5 and 8 MHz. Ultrasound elevated the expression of early response genes c-Fos, c-Jun and c-Myc, maximized at 5 MHz. The phosphorylated ERK inhibitor PD98059 abrogated any increase in c-series gene expression, suggesting that signaling occurs via the MAPPK/ERK pathway. However, phosphorylated ERK levels did not change with ultrasound frequency, indicating that processes downstream of ERK phosphorylation (such as nuclear transport and chromatin reorganization) respond to ultrasound with frequency dependence. A quantitative, biphasic mathematical model based on Biot theory predicted that cytoplasmic and nuclear stress is maximized at 5.2 ± 0.8 MHz for a chondrocyte, confirming experimental measurements.

Glioblastoma is the most aggressive and deadly brain cancer. There is growing interest to develop drugs that specifically target to glioblastoma tumor-initiating cells (TICs). However, the cost-effective production of large numbers of high quality glioblastoma TICs for drug discovery with current cell culturing technologies remains very challenging. Here, we report a new method that cultures glioblastoma TICs in microscale alginate hydrogel tubes (or AlgTubes). The AlgTubes allowed long-term culturing (~50 days, 10 passages) of glioblastoma TICs with high growth rate (~700-fold expansion/14 days), high cell viability and high volumetric yield (~3.0 × 108 cells/mL) without losing the stem cell properties, all offered large advancements over current culturing methods. This method can be applied for the scalable production of glioblastoma TICs at affordable cost for drug discovery.