A novel pathogen detection system based on high-resolution CE-SSCP using a triblock copolymer matrix.

Although CE-SSCP analysis combined with 16S ribosomal RNA gene-specific PCR has enormous potential as a simple and versatile pathogen detection technique, low resolution of CE-SSCP causes the limited application. Among the experimental conditions affecting the resolution, the polymer matrix is considered to be most critical to improve the resolution of CE-SSCP analysis. However, due to the peak broadening caused by the interaction between hydrophobic moiety of polymer matrices and DNA, conventional polymer matrices are not ideal for CE-SSCP analysis. A poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) triblock copolymer, with dynamic coating ability and a propensity to form micelles to minimize exposure of hydrophobic PPO block to DNA, can be an alternative matrix. In this study, we examined the resolution of CE-SSCP analysis using the PEO-PPO-PEO triblock copolymer as the polymer matrix and four same-sized DNA fragments of similar sequence content. Among 48 commercially available PEO-PPO-PEO triblock copolymers, three were selected due to their transparency in the operable range of viscosity and PEO(137)PPO(43)PEO(137) exhibited the most effective separation. Significant improvement in resolution allowed discrimination of the similar sequences, thus greatly facilitated CE-SSCP analysis compared to the conventional polymer matrix. The results indicate that PEO-PPO-PEO triblock copolymer may serve as an ideal matrix for high-resolution CE-SSCP analysis.

Cardiac response to startle stimuli in larval zebrafish: sympathetic and parasympathetic components.

Central regulation of cardiac output via the sympathetic and parasympathetic branches of the autonomic nervous system allows the organism to respond to environmental changes. Sudden onset stimuli, startle stimuli, are useful probes to study central regulatory responses to the environment. In mammals, startle stimuli induce a transient bradycardia that habituates with repeated stimulation. Repeated presentation of the stimulus results in tachycardia. In this study, we investigate the behavioral regulation of heart rate in response to sudden stimuli in the zebrafish. Larval zebrafish show a stereotyped heart rate response to mild electrical shock. Naïve fish show a significant increase in interbeat interval that resolves in the 2 s following stimulation. This transient bradycardia decreases on repeated exposure to the stimulus. Following repeated stimulation, the fish become tachycardic within 1 min of stimulation. Both the transient bradycardia and following tachycardia responses are blocked with administration of the ganglionic blocker hexamethonium, demonstrating that these responses are mediated centrally. The transient bradycardia is blocked by the muscarinic antagonist atropine, suggesting that this response is mediated by the parasympathetic system, while the following tachycardia is specifically blocked by the beta-adrenergic antagonist propranolol, suggesting that this response is mediated by the sympathetic nervous system. Together, these results demonstrate that at the larval stage, zebrafish actively regulate cardiac output to changes in their environment using both the parasympathetic and sympathetic branches of the autonomic nervous system, a behavioral response that is markedly similar to that observed in mammals to similar sudden onset stimuli.