Persistence of a Stx-Encoding Bacteriophage in Minced Meat Investigated by Application of an Improved DNA Extraction Method and Digital Droplet PCR.

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens with Shiga toxins as the main virulence factor. Shiga toxins are encoded on Shiga toxin-encoding bacteriophages (Stx phages). Stx phages may exist as free bacteriophages in the environment or in foods or as prophages integrated into the host genome. From a food safety perspective, it is important to have knowledge on the survival and persistence of Stx phages in food products since these may integrate into the bacterial hosts through transduction if conditions are right. Here, we present the results from a study investigating the survival of a Stx phage in minced meat from beef stored at a suboptimal temperature (8°C) for food storage along with modifications and optimizations of the methods applied. Minced meat from beef was inoculated with known levels of a labeled Stx phage prior to storage. Phage filtrates were used for plaque assays and DNA extraction, followed by real-time PCR and digital droplet PCR (ddPCR). The results from the pilot study suggested that the initial DNA extraction protocol was not optimal, and several modifications were tested before a final protocol was defined. The final DNA extraction protocol comprised ultra-centrifugation of the entire phage filtrate for concentrating phages and two times phenol-chloroform extraction. The protocol was used for two spiking experiments. The DNA extraction protocol resulted in flexibility in the amount of DNA available for use in PCR analyses, ultimately increasing the sensitivity of the method used for quantification of phages in a sample. All three quantification methods employed (i.e., plaque assays, real-time PCR, and ddPCR) showed similar trends in the development of the phages during storage, where ddPCR has the benefit of giving absolute quantification of DNA copies in a simple experimental setup. The results indicate that the Stx phages persist and remain infective for at least 20 days under the storage conditions used in the present study. Stx phages in foods might represent a potential risk for humans. Although it can be speculated that transduction may take place at 8°C with subsequent forming of STEC, it can be expected to be a rare event. However, such an event may possibly take place under more optimal conditions, such as an increase in storage temperature of foods or in the gastrointestinal tract of humans.

Mechanisms involved in alternariol-induced cell cycle arrest.

Alternariol (AOH), a mycotoxin produced by Alternaria sp, is often found as a contaminant in fruit and cereal products. Here we employed the murine macrophage cell line RAW 264.7 to test the hypothesis that AOH causes toxicity as a response to DNA damage. AOH at concentrations of 15-30µM almost completely blocked cell proliferation. Within 30min treatment, AOH (30µM) significantly increased the level of reactive oxygen species (ROS). Furthermore, DNA base oxidations as well as DNA strand breaks and/or alkaline labile sites were detected by the comet assay after 2h exposure of AOH. Cell death (mostly necrosis) was observed after prolonged exposure to the highest concentration of AOH (60µM for 24 and 48h) in our study. The DNA damage response involved phosphorylation (activation) of histone H2AX and check point kinase-1- and 2 (Chk-1/2). Moreover, AOH activated p53 and increased the expression of p21, Cyclin B, MDM2, and Sestrin 2; likewise the level of several miRNA was affected. AOH-induced Sestrin 2 expression was regulated by p53 and could at least partly be inhibited by antioxidants, suggesting a role of ROS in the response. Interestingly, the addition of antioxidants did not inhibit cell cycle arrest. Although the formation of ROS by itself was not directly linked cell proliferation, AOH-induced DNA damage and resulting transcriptional changes in p21, MDM2, and Cyclin B likely contribute to the reduced cell proliferation; while Sestrin 2 would contribute to the oxidant defense.

Reduced [3H]IP3 binding but unchanged IP3 receptor levels in the rat hippocampus CA1 region following transient global ischemia and tolerance induction.

Changes in inositol (1,4,5)-trisphosphate (IP3) binding properties and the protein level of the IP3 receptor have been reported in different pathological conditions in the brain, e.g. cerebral ischemia, Alzheimer's disease, and Huntingtons disease. We used the 4-vessel occlusion model in rat brain to investigate the effect of transient ischemia insults on the IP3 receptor mRNA level, the IP3 receptor protein level and [3H]IP3 binding. Recirculation periods were limited (1-72 h) to avoid the development of delayed neuronal death. We found that the IP3 receptor mRNA levels were decreased after damage-inducing ischemia (9 min) in the hippocampus CA1 and CA3 regions. The mRNA levels were unaltered after tolerance-inducing ischemia (3 min). However, [3H]IP3 binding was significantly reduced after both damage- and tolerance-inducing ischemia in the hippocampus CA1 region. Furthermore, all investigated brain areas showed a decreased [3H]IP3 binding when tolerance-inducing ischemia was followed by a second ischemic insult (3 + 8.5 min ischemia). The IP3 receptor protein levels remained constant in all investigated brain areas. These results indicate that a reduced [3H]IP3 binding capability in the particularly vulnerable areas occurs as an early consequence of cerebral ischemia, before IP3 receptor protein levels are reduced in these areas. Structural or conformational changes altering IP3 binding may be of necessity on the pathway leading to down-regulation of IP3 receptor protein levels, as observed by others.