Positive correlation between the generation of reactive oxygen species and activation/reactivation of transgene expression after hydrodynamic injections into mice.

Purpose : Hydrodynamic injection has been shown to reactivate silenced transgene expression in mouse liver. In this study, the roles of inflammatory cytokines and reactive oxygen species (ROS) in the reactivation were examined. Methods : Production of inflammatory cytokines and ROS by hydrodynamic injection of saline was examined in mice that had received a hydrodynamic injection of a plasmid expressing Gaussia luciferase. The level of reporter gene expression was used as an indicator of the reactivation. The involvement of cytokines and ROS was examined by depleting Kupffer cells or by pre-administration of antioxidants, respectively. Results : A hydrodynamic injection of saline induced a significant production of interleukin (IL)-6. Depleting Kupffer cells using clodronate liposomes markedly reduced the IL-6 production but had no significant effect on the transgene expression. On the other hand, an injection of catalase or N-acetylcysteine significantly inhibited the hydrodynamic injection-induced reactivation of silenced transgene expression. The silenced expression was also reactivated by carbon tetrachloride, an inducer of oxidative stress in the liver, in a dose-dependent manner, and this reactivation was significantly inhibited by catalase. Conclusions : These findings show a positive correlation between the generation of ROS and the reactivation of silenced transgene expression after hydrodynamic injections

A stochastic assessment of the public health risks of fluoroquinolone resistance Campylobacter and the use of the drug in broiler production in Japan

Purpose: Antimicrobial resistance (AMR) is a major global public health and a food safety issue. Risk analysis is an essential tool in assessing the risk to human health from foodborne AMR microorganisms and determining appropriate risk management strategies to control those risks. Although quantitative risk assessments are encouraged and have been performed in some EU countries and the US, so far only qualitative risk assessments are performed in Japan. This project was commissioned by the Japan Food Safety Commission in the context of its programme to develop a quantitative risk assessment, with Campylobacter in broiler and use of fluoroquinolone (FQ) for broiler production as an example. Methods: The risk assessment model was subdivided into the release, exposure, and consequence assessment sections. In the release section, FQ resistance mechanism and data from Japanese Veterinary Antimicrobial Resistance Monitoring Program were utilized for the model development. The model output was percentage of FQ resistance Campylobacter (FRC) among broiler borne human campylobacteriosis. In order to take uncertainties in the data into account, the model was built, and simulations were performed using @Risk 4.5 (Palisade Corp.) . Results: The average percentage of broilers infected with FRC was estimated to be 34.0%. Among FRC strains, 10.4% carried a mutation at Thr86Ile of gyrA with overexpression producing strains of efflux pump, 20.8% carried a same mutation of gyrA with low expression producing strains of efflux pump, and 2.8% carried a mutation of GyrA other places than Thr86Ile. The estimated that an average and the maximum annual number of Campylobacter infections per person were 1.02, and 88 times, respectively. Conclusions: In the maximum (88 infections per year) scenario, the patient is estimated to be infected with FRC 30 times, and 28 times out of 30 times, he or she is estimated to be infected with Campylobacter with a mutation of Thr86Ile of GyrA. Relevance: By using this model, use of FQ in the farm and the effect on the percentage of FRC in broilers was not identified. We couldn't find any adverse human health effects associated with FRC strains

Near-Infrared Fluorescence Probes for Enzymes Based on Binding Affinity Modulation of Squarylium Dye Scaffold

We present a novel design strategy for near-infrared (NIR) fluorescence probes utilizing dye–protein interaction as a trigger for fluorescence enhancement. The design principle involves modification of a polymethine dye with cleavable functional groups that reduce the dye’s protein-binding affinity. When these functional groups are removed by specific interaction with the target enzymes, the dye’s protein affinity is restored, protein binding occurs, and the dye’s fluorescence is strongly enhanced. To validate this strategy, we first designed and synthesized an alkaline phosphatase (ALP) sensor by introducing phosphate into the squarylium dye scaffold; this sensor was able to detect ALP-labeled secondary antibodies in Western blotting analysis. Second, we synthesized a probe for β-galactosidase (widely used as a reporter of gene expression) by means of β-galactosyl substitution of the squarylium scaffold; this sensor was able to visualize β-galactosidase activity both in vitro and in vivo. Our strategy should be applicable to obtain NIR fluorescence probes for a wide range of target enzymes