Single-molecule binding characterization of primosomal protein PriA involved in replication restart.

DNA damage leads to stalled or collapsed replication forks. Replication restart primosomes re-initiate DNA synthesis at these stalled or collapsed DNA replication forks, which is important for bacterial survival. Primosomal protein PriA specifically recognizes the DNA fork structure and recruits other primosomal proteins to load the replicative helicase, in order to re-establish the replication fork. PriA binding on DNA is the first step to restart replication forks for proper DNA repair. Using a single-molecule fluorescence colocalization experiment, we measured the thermodynamic and real-time kinetic properties of fluorescence-labeled Gram-positive bacteria Geobacillus stearothermophilus PriA binding on DNA forks. We showed that PriA preferentially binds to a DNA fork structure with a fully duplexed leading strand at sub-nanomolar affinity (Kd = 268 ± 99 pM). PriA binds dynamically, and its association and dissociation rate constants can be determined using the appearance and disappearance of the fluorescence signal. In addition, we showed that PriA binds to DNA forks as a monomer using photobleaching step counting. This information offers a molecular basis essential for understanding the mechanism of replication restart.

Crosstalk between CST and RPA regulates RAD51 activity during replication stress.

Replication stress causes replication fork stalling, resulting in an accumulation of single-stranded DNA (ssDNA). Replication protein A (RPA) and CTC1-STN1-TEN1 (CST) complex bind ssDNA and are found at stalled forks, where they regulate RAD51 recruitment and foci formation in vivo. Here, we investigate crosstalk between RPA, CST, and RAD51. We show that CST and RPA localize in close proximity in cells. Although CST stably binds to ssDNA with a high affinity at low ionic strength, the interaction becomes more dynamic and enables facilitated dissociation at high ionic strength. CST can coexist with RPA on the same ssDNA and target RAD51 to RPA-coated ssDNA. Notably, whereas RPA-coated ssDNA inhibits RAD51 activity, RAD51 can assemble a functional filament and exhibit strand-exchange activity on CST-coated ssDNA at high ionic strength. Our findings provide mechanistic insights into how CST targets and tethers RAD51 to RPA-coated ssDNA in response to replication stress.

Effect of Viewing Angle on Volume Perceptions for Paired Tumblers.

This study examined how tumbler characteristics influenced the perception of volume at different viewing angles. Three tumbler characteristics were individually examined, namely, shape, size, and elongation. At four viewing angles (0°, 30°, 60°, and 90°), 50 participants poured a certain amount of liquid (150 or 200 mL) into a designated tumbler according to their perception. Results showed that tumbler size and elongation influenced volume perception. At viewing angles of 0° and 30°, the participants poured more liquid into short-wide tumblers than into tall-slender tumblers. At viewing angles of 60° and 90°, the results were opposite. The reason may be that the change of viewing angle made the participant's sight cues from the container diameter more visible than those from the container height. Similar results were obtained for the pair of small and large tumblers. However, no effect of viewing angle on tumblers with different geometric shapes was observed. The contradictory results in comparison with those of previous studies may be related to viewing angle; in addition, the effect of viewing angle was also influenced by the characteristics of tumblers.

Physical characterizations and sustained release profiling of gastroretentive drug delivery systems with improved floating and swelling capabilities.

The aim was to develop gastroretentive drug delivery systems (GRDDSs) by combining floating and swelling. GRDDS tablets formulated with hydroxyethylcellulose (HEC), chitosan (CS) and sodium bicarbonate (SB) for evaluating floating capacity (floating lag time and duration) and swelling characteristics. CS was used because it was swellable in acidic media and biocompatible. Losartan was incorporated into the optimized formulations for sustained release profiling. Results demonstrated that for those formulations at HEC:CS ratio of 5:5 containing CS, both the floating lag time and floating duration were optimal and reached the preferred swelling effect and sustain for 24h. Adding SB improved the floating capabilities for all ratios of HEC:CS, but reduced the swelling ability for those formulations containing a higher portion of low viscosity grade CS. Sustained release profiles for losartan in those formulations were achievable, using all viscosity grades of CS at all examined HEC:CS ratios; however, it is more adjustable at different HEC:CS ratios when using a lower viscosity grade of CS. Optimized GRDDS formulations for losartan composed of an equivalent ratio of HEC to CS with 20mg SB resulted in the tablets floating for more than 16 h and an adjustable sustained release profile.