Functional analysis of the aefR mutation and identification of its binding site in Pseudomonas syringae pv. tabaci 11528.

The TetR family transcriptional regulator AefR contributes to the regulation of the quorum-sensing system. However, the role of AefR in the regulatory network of the phytopathogen Pseudomonas syringae pathovars is not known. In this study, the phenotype of a P. syringae pv. tabaci 11528 aefR deletion mutant strain was examined. The aefR gene expression and AefR DNA-binding affinity were examined by quantitative real-time polymerase chain reaction and electrophoretic mobility shift assay, respectively. AefR was found to control quorum-sensing genes as well as the efflux genes mexE, mexF, and oprN via an indirect mechanism. AefR binds to its own operator site as well as to the palindromic sequence between positions -28 and -2 corresponding to the transcription start site of aefR, as determined by dye primer sequencing. These results suggest that P. syringae AefR modulates quorum sensing and efflux as well as its own expression, which can be exploited by strategies developed to manage this plant parasite.

Diversity and homogeneity in responses of midbrain dopamine neurons.

Dopamine neurons of the ventral midbrain have been found to signal a reward prediction error that can mediate positive reinforcement. Despite the demonstration of modest diversity at the cellular and molecular levels, there has been little analysis of response diversity in behaving animals. Here we examine response diversity in rhesus macaques to appetitive, aversive, and neutral stimuli having relative motivational values that were measured and controlled through a choice task. First, consistent with previous studies, we observed a continuum of response variability and an apparent absence of distinct clusters in scatter plots, suggesting a lack of statistically discrete subpopulations of neurons. Second, we found that a group of "sensitive" neurons tend to be more strongly suppressed by a variety of stimuli and to be more strongly activated by juice. Third, neurons in the "ventral tier" of substantia nigra were found to have greater suppression, and a subset of these had higher baseline firing rates and late "rebound" activation after suppression. These neurons could belong to a previously identified subgroup of dopamine neurons that express high levels of H-type cation channels but lack calbindin. Fourth, neurons further rostral exhibited greater suppression. Fifth, although we observed weak activation of some neurons by aversive stimuli, this was not associated with their aversiveness. In conclusion, we find a diversity of response properties, distributed along a continuum, within what may be a single functional population of neurons signaling reward prediction error.

Multiphasic temporal dynamics in responses of midbrain dopamine neurons to appetitive and aversive stimuli.

The transient response of dopamine neurons has been described as reward prediction error (RPE), with activation or suppression by events that are better or worse than expected, respectively. However, at least a minority of neurons are activated by aversive or high-intensity stimuli, casting doubt on the generality of RPE in describing the dopamine signal. To overcome limitations of previous studies, we studied neuronal responses to a wider variety of high-intensity and aversive stimuli, and we quantified and controlled aversiveness through a choice task in which macaques sacrificed juice to avoid aversive stimuli. Whereas most previous work has portrayed the RPE as a single impulse or "phase," here we demonstrate its multiphasic temporal dynamics. Aversive or high-intensity stimuli evoked a triphasic sequence of activation-suppression-activation extending over a period of 40-700 ms. The initial activation at short latencies (40-120 ms) reflected sensory intensity. The influence of motivational value became dominant between 150 and 250 ms, with activation in the case of appetitive stimuli, and suppression in the case of aversive and neutral stimuli. The previously unreported late activation appeared to be a modest "rebound" after strong suppression. Similarly, strong activation by reward was often followed by suppression. We suggest that these "rebounds" may result from overcompensation by homeostatic mechanisms in some cells. Our results are consistent with a realistic RPE, which evolves over time through a dynamic balance of excitation and inhibition.

Synthesis of licochalcone analogues with increased anti-inflammatory activity.

Licohalcones have been reported to have various biological activities. However, most of licochalcones also showed cytotoxicity even though their versitile utilities. Licochalcones B and D, which have common substituents at aromatic ring B, are targeted to modify the structure at aromatic ring A for inflammatory studies. Licochalcone derivatives (1-6) thus prepared are compared for their suppression ability of nitric oxide (NO) production and showed 9.94, 4.72, 10.1, 4.85, 2.37 and 4.95µM of IC50 values, respectively.

Comparison of different methods of RNA preparation from peripheral blood for nucleic acid amplification assay.

BACKGROUND: Nucleic acid amplification assays (NAAs), such as polymerase chain reaction or loop-mediated isothermal amplification (LAMP), are used for disease diagnosis. Current nucleic acid isolation kits require several hours for completion of protocol including the complicated handling steps. OBJECTIVE: In this study, a simple and cost-effective nucleic acid preparation method was developed, and its performance was compared with those of commercial kits. MATERIALS AND METHODS: RNA was prepared using our method and three commercial RNA isolation kits. The RNA quantity and quality were evaluated using the NanoDrop spectrophotometer and Agilent 2100 bioanalyser. Reverse transcription LAMP (RT-LAMP) reactions were performed to determine the usability of the RNA preparation methods. RESULTS: The concentrations of RNA extracted from blood samples by four different methods were sufficient for use in NAAs. The RNA integrity number was> 7.0 when RNA was isolated using other RNA isolation kits but lower when prepared using our method. The RT-LAMP reaction was successfully performed when RNA was prepared using any of the methods. CONCLUSIONS: These results demonstrate that despite the lower purity and integrity of RNA, our RNA preparation protocol is simple and rapid and shows reasonable performance in RT-LAMP.