All-atom molecular dynamics study of hepatitis B virus containing pregenome RNA in solution.

Immature hepatitis B virus (HBV) captures nucleotides in its capsid for reverse transcription. The nucleotides and nucleotide analog drugs, which are triphosphorylated and negatively charged in the cell, approach the capsid via diffusion and are absorbed into it. In this study, we performed a long-time molecular dynamics calculation of the entire HBV capsid containing pregenome RNA to investigate the interactions between the capsid and negatively charged substances. Electric field analysis demonstrated that negatively charged substances can approach the HBV capsid by thermal motion, avoiding spikes. The substances then migrate all over the floor of the HBV capsid. Finally, they find pores through which they can pass through the HBV capsid shell. Free energy profiles were calculated along these pores for small ions to understand their permeability through the pores. Anions (Cl-) showed higher free energy barriers than cations (Na+ and K+) through all pores, and the permeation rate of Cl- was eight times slower than that of K+ or Na+. Furthermore, the ions were more stable in the capsid than in the bulk water. Thus, the HBV capsid exerts ion selectivity for uptake and provides an environment for ions, such as nucleotides and nucleotide analog drugs, to be stabilized within the capsid.

Installation of multiple automated external defibrillators to prevent sudden death in school-aged children.

BACKGROUND: Recently, a student died of idiopathic ventricular fibrillation in a school where an automated external defibrillator (AED) had been installed. The tragedy could not be prevented because the only AED in the school was installed in the teachers' office, far from the school ground where the accident took place. This prompted establishment of a multiple AED system in schools. The aim of this study was to analyze the efficacy of the multiple AED system to prevent sudden death in school-aged children. METHODS: Assumed accident sites consisted of the school ground, gymnasium, Judo and Kendo hall, swimming pool, and classrooms on the first and the fourth floor. Multiple AED were installed in the teachers' office, gymnasium, some classrooms, and also provided as a portable AED in a rucksack. The time from the accident site to the teachers' office for single AED, and from the accident site to the nearest AED for multiple AED, was calculated. RESULTS: The AED retrieval time was significantly shorter in 55 elementary schools and in 29 junior high schools when multiple AED were installed compared with single AED. Except for the classroom on the fourth floor, the number of people who took >120 s to bring the AED to the accident site was lower when multiple AED were installed compared with the single AED. CONCLUSION: Multiple AED provided in appropriate sites can reduce the time to reach the casualty and hence prevent sudden death in school-aged children.

Three-dimensional invasion of epithelial-mesenchymal transition-positive human peritoneal mesothelial cells into collagen gel is promoted by the concentration gradient of fibronectin.

BACKGROUND: In long-term peritoneal dialysis, myofibroblast-like cells found in the interstitium of the peritoneum are assumed to be a transformed type of mesothelial cell-epithelial-mesenchymal transition-positive [EMT(+)] human peritoneal mesothelial cells (HPMCs)-because they express a mesothelial marker, cytokeratin. However, no direct evidence about how these cells are able to invade from the mesothelium has yet been obtained. AIM: In this study, we aimed to verify whether EMT(+) HPMCs would, in vitro, invade three-dimensionally along certain chemotactic factors. METHODS: We used reverse-transcriptase polymerase chain reaction to measure expression of Snail, E-cadherin, α(5)-integrin, and matrix metalloproteinase 2 (MMP2) messenger RNA (mRNA) in HPMCs exposed to 10 ng/mL transforming growth factor ß1 (TGFß1) and how that expression corresponds to cell motility, as represented by a video movie. We used the Transwell (12 µm pore diameter: Sigma-Aldrich, Tokyo, Japan) to construct a three-dimensional (3D) cell migration chamber. In the lower chamber, a concentration gradient of fibronectin (FN) or albumin(Alb) was formed in 0.1% type I collagen by diffusion (C(0)=22 nmol/L; concentration gradient: C/C(0)=0.7). All cells beneath the membrane were counted 72 hours after 5×10(4) EMT(+) HPMCs (HPMCs after a 48-hour exposure to 10 ng/mL TGFß1) had been spread in the upper chamber. RESULTS: After 72 hours, the increased motility of HPMCs resulting from their exposure to 10 ng/mL TGFß1 had returned to baseline, but they retained an elongated morphology. Expression of Snail and MMP2 mRNA reached maximum at 24 hours. Expression of E-cadherin declined, and expression of α(5)-integrin increased continuously. In the 3D invasion study, significantly enhanced invasion by EMT(+) but not EMT(-) HPMCs was clearly seen in the presence of a FN concentration gradient (p<0.01), although invasion by EMT(+) and EMT(-) HPMCs in the absence of a FN concentration gradient was not statistically significantly different. Compared with the EMT(+) control (no concentration gradient), invasion by EMT(+) HPMCs was 2.1 ± 0.5 times (p<0.05) and 1.4 ± 0.4 times (p=nonsignificant) higher along the FN and Alb concentration gradients respectively. Increased invasion along the FN concentration gradient was significantly inhibited (p<0.05) when the HPMCs were pre-incubated with 5 µg/mL RGDS (a blocker for α(5)-integrin to FN). CONCLUSIONS: We conclude that EMT(+) HPMCs invade collagen gel along the FN concentration gradient because of specific binding to RGDS receptors, which bind integrins such as α(5)-integrin, upregulating invasion-related gene expression associated with synthesis of the cytoskeleton protein α smooth muscle actin.