Development of a loop-mediated Isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus

<p>Abstract</p> <p>Background</p> <p><it>Vibrio parahaemolyticus </it>is a marine seafood-borne pathogen causing gastrointestinal disorders in humans. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of <it>V. parahaemolyticus</it>. Most <it>V. parahaemolyticus </it>isolates from the environment do not produce TDH or TRH. Total <it>V. parahaemolyticus </it>has been used as an indicator for control of seafood contamination toward prevention of infection. Detection of total <it>V. parahaemolyticus </it>using conventional culture- and biochemical-based assays is time-consuming and laborious, requiring more than three days. Thus, we developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the sensitive and rapid detection of <it>Vibrio parahaemolyticus</it>.</p> <p>Results</p> <p>The assay provided markedly more sensitive and rapid detection of <it>V. parahaemolyticus </it>strains than conventional biochemical and PCR assays. The assay correctly identified 143 <it>V. parahaemolyticus </it>strains, but did not detect 33 non-<it>parahaemolyticus Vibrio </it>and 56 non-<it>Vibrio </it>strains. Sensitivity of the LAMP assay for direct detection of <it>V. parahaemolyticus </it>in pure cultures and in spiked shrimp samples was 5.3 × 10²CFU per ml/g (2.0 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13–22 min in a single colony on TCBS agar from each of 143 <it>V. parahaemolyticus </it>strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of <it>V. parahaemolyticus </it>required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination.</p> <p>Conclusion</p> <p>The LAMP assay is a sensitive, rapid and simple tool for the detection of <it>V. parahaemolyticus </it>and will facilitate the surveillance for control of contamination of <it>V. parahaemolyticus </it>in seafood.</p

Effects of Sulfonylureas on Periodontopathic Bacteria-Induced Inflammation.

福岡歯科大学2019年

Glyburide inhibits the bone resorption induced by traumatic occlusion in rats.

To examine whether glyburide inhibits bone destruction caused by traumatic occlusion in a rat occlusal trauma model.Excessive mechanical stress, such as traumatic occlusion, induces expression of IL-1β and may be involved in bone resorption. NLRP3 inflammasomes have been linked to IL-1β expression, but it is currently unclear whether glyburide, the inhibiter of NLRP3 inflammasome, suppresses occlusal trauma in rats.Male SD rats aged 7 weeks were used. In the trauma group, the occlusal surface of the maxillary first right molar was raised by attaching a metal wire to apply occlusal trauma to the mandibular first right molar. In the trauma + glyburide group, the NLRP3 inhibitor glyburide was administered orally every 24 hours from 1 day before induction of occlusal trauma. Rats were euthanized after 5 or 10 days, and the maxillary first molars were harvested with the adjacent tissues for histopathological investigation. Immunohistochemical expression of IL-1β, NLRP3, and RANKL was also assessed.On day 5, bone resorption was significantly greater in the trauma group compared with the control group or the trauma + glyburide group, and there were significantly higher numbers of osteoclasts and cells positive for IL-1β, NLRP3, and RANKL in the trauma group.In this study, glyburide inhibits bone resorption by traumatic occlusion in rats. It suggests that the NLRP3/IL-1β pathway might be associated with bone resorption induced by traumatic occlusion.福岡歯科大学2019年

Thymidine Catabolism as a Metabolic Strategy for Cancer Survival

Thymidine phosphorylase (TP), a rate-limiting enzyme in thymidine catabolism, plays a pivotal role in tumor progression; however, the mechanisms underlying this role are not fully understood. Here, we found that TP-mediated thymidine catabolism could supply the carbon source in the glycolytic pathway and thus contribute to cell survival under conditions of nutrient deprivation. In TP-expressing cells, thymidine was converted to metabolites, including glucose 6-phosphate, lactate, 5-phospho-α-D-ribose 1-diphosphate, and serine, via the glycolytic pathway both in vitro and in vivo. These thymidine-derived metabolites were required for the survival of cells under low-glucose conditions. Furthermore, activation of thymidine catabolism was observed in human gastric cancer. These findings demonstrate that thymidine can serve as a glycolytic pathway substrate in human cancer cells

Thymidine catabolism promotes NADPH oxidase-derived reactive oxygen species (ROS) signalling in KB and yumoto cells

Thymidine phosphorylase (TP) is a rate-limiting enzyme in the thymidine catabolic pathway. TP is identical to platelet-derived endothelial cell growth factor and contributes to tumour angiogenesis. TP induces the generation of reactive oxygen species (ROS) and enhances the expression of oxidative stress-responsive genes, such as interleukin (IL)-8. However, the mechanism underlying ROS induction by TP remains unclear. In the present study, we demonstrated that TP promotes NADPH oxidase-derived ROS signalling in cancer cells. NADPH oxidase inhibition using apocynin or small interfering RNAs (siRNAs) abrogated the induction of IL-8 and ROS in TP-expressing cancer cells. Meanwhile, thymidine catabolism induced by TP increased the levels of NADPH and intermediates of the pentose phosphate pathway (PPP). Both siRNA knockdown of glucose 6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme in PPP, and a G6PD inhibitor, dihydroepiandrosterone, reduced TP-induced ROS production. siRNA downregulation of 2-deoxy-D-ribose 5-phosphate (DR5P) aldolase, which is needed for DR5P to enter glycolysis, also suppressed the induction of NADPH and IL-8 in TP-expressing cells. These results suggested that TP-mediated thymidine catabolism increases the intracellular NADPH level via the PPP, which enhances the production of ROS by NADPH oxidase and activates its downstream signalling

A dehydrated space-weathered skin cloaking the hydrated interior of Ryugu

Without a protective atmosphere, space-exposed surfaces of airless Solar System bodies gradually experience an alteration in composition, structure and optical properties through a collective process called space weathering. The return of samples from near-Earth asteroid (162173) Ryugu by Hayabusa2 provides the first opportunity for laboratory study of space-weathering signatures on the most abundant type of inner solar system body: a C-type asteroid, composed of materials largely unchanged since the formation of the Solar System. Weathered Ryugu grains show areas of surface amorphization and partial melting of phyllosilicates, in which reduction from Fe3+ to Fe2+ and dehydration developed. Space weathering probably contributed to dehydration by dehydroxylation of Ryugu surface phyllosilicates that had already lost interlayer water molecules and to weakening of the 2.7 µm hydroxyl (–OH) band in reflectance spectra. For C-type asteroids in general, this indicates that a weak 2.7 µm band can signify space-weathering-induced surface dehydration, rather than bulk volatile loss

Characterization of Third-Generation-Cephalosporin-Resistant Shiga Toxin-Producing Strains of Escherichia coli O157:H7 in Japan

We isolated Shiga toxin-producing Escherichia coli O157:H7 strains resistant to third-generation cephalosporins. The resistant strains harbored bla CMY-2, a plasmid-mediated AmpC β-lactamase. Genotyping of isolates revealed the possible spread of this problematic bacterium. Results suggested the importance of the investigation and surveillance of enterobacteria with plasmids harboring bla CMY-2