Aims: To evaluate the suitability of using sterile water and phosphate-buffered saline (PBS) for preservation of bacteria pathogenic to plants or humans. Methods and Results: The stationary-phase bacterial cells collected from rich agar media were transferred to 10 ml of sterile water or PBS (pH 7AE2) containing KH 2 PO 4 , 15AE44 lM M; NaCl, 1AE55 mM M; Na 2 HPO 4 , 27AE09 lM M in a screw-cap tube. The tubes were sealed with parafilm membranes and stored in the dark and at room temperature. Almost all the bacteria tested (148 strains), including Pseudomonas fluorescens, P. viridiflava, Erwinia spp., Xanthomonas campestris, Cytophaga johnsonae, Salmonella spp., Yersinia enterocolitica, Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus, survived in water for at least several months and up to 16 years. A vast majority of the Gram-negative bacteria tested survived equally well in water and in PBS for at least 30 weeks. However, the populations of two Gram-positive bacteria [G(+)], L. monocytogenes and Staph. aureus, declined more rapidly in water than in PBS. Conclusions: Plant-and human-pathogenic bacteria can be preserved in pure water or PBS for several years. G(+) bacteria appear to survive better in PBS than in water. Significance and Impact of the Study: The method described here is a simple and economical means for preservation of bacterial cultures, which is especially useful for laboratories not equipped with the lyophilizer or ultra-low freezer. Long-term survival of food-borne pathogens in water underlines the importance of water as a potential vehicle for transmitting the diseases.

This study clarifies the mechanism by which naked DNA is eliminated from the circulation after i.v. injection, focusing on the role of both the liver and blood compartments in vivo (i.e. mouse). With this knowledge, we can more clearly understand the mechanism of naked DNA clearance and develop more efficient strategies for DNA transfer in vivo.

The vectors for gene delivery are usually classified as viral and nonviral vectors. While the viral vectors are very efficient in transducing cells, safety concerns regarding their use in humans make nonviral vectors an attractive alternative. Among the nonviral vectors, the lipoplexes (complexes of cationic liposome/pDNA) are the most studied and represent the most promising approaches for human clinical trials. However, an inflammatory response is invariably associated with administration of the lipoplexes, which must be avoided in the clinical application. Here, we have successfully developed a nonimmunostimulatory vector for gene therapy. The vector possesses dual functions of: 1) efficiently delivering a gene to target cells and 2) codelivering DNA and inflammatory suppressors into the immune cells where the released suppressor can inhibit cytokine production. The inflammatory suppressors successfully delivered by the vector included glucocorticoids, a nonsteroidal anti-inflammatory drug (NSAID), an NF-kappaB inhibitor, and a natural compound from an herbal medicine. Intravenous injection of the vector dramatically suppressed the cytokine production induced by CpG motif pDNA, including TNF-alpha, IL-12 and IFN-gamma. This new gene vector has a great potential in clinical gene therapy. Another potential use of the vector is codelivery of an enhancer candidate, acting at the transcriptional and translational levels to improve the efficiency of gene transfer by the nonviral vector. Moreover, the unique feature of this vector is that it can be used as an easy and powerful tool for in vivo screening of anti-inflammatory drugs.

This study provides new insights into the mechanism of electro-gene transfer, and may allow the definition of newer and more efficient conditions for in vivo electroporation.