Efficient DNA Extraction from Nail Clippings Using the Protease Solution from <Emphasis Type="Italic">Cucumis melo</Emphasis>

Owing to the increasing importance of genomic information, obtaining genomic DNA easily from biological specimens has become more and more important. This article proposes an efficient method for obtaining genomic DNA from nail clippings. Nail clippings can be easily obtained, are thermostable and easy to transport, and have low infectivity. The drawback of their use, however, has been the difficulty of extracting genomic material from them. We have overcome this obstacle using the protease solution obtained from Cucumis melo. The keratinolytic activity of the protease solution was 1.78-fold higher than that of proteinase K, which is commonly used to degrade keratin. With the protease solution, three times more DNA was extracted than when proteinase K was used. In order to verify the integrity of the extracted DNA, genotype analysis on 170 subjects was performed by both PCR–RFLP and Real Time PCR. The results of the genotyping showed that the extracted DNA was suitable for genotyping analysis. In conclusion, we have developed an efficient extraction method for using nail clippings as a genome source and a research tool in molecular epidemiology, medical diagnostics, and forensic science.     

VP2118 has major roles in Vibrio parahaemolyticus response to oxidative stress

Background

Reactive oxygen species (ROS), including superoxide anion radical, induce chronic risk of oxidative damage to many cellular macromolecules resulting in damage to cells. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vibrio parahaemolyticus, a marine bacterium that causes acute gastroenteritis following consumption of raw or undercooked seafood, can survive ROS generated by intestinal inflammatory cells. However, there is little information concerning SODs in V. parahaemolyticus. This study aims to clarify the role of V. parahaemolyticus SODs against ROS.

Methods

V. parahaemolyticus SOD gene promoter activities were measured by a GFP reporter assay. Mutants of V. parahaemolyticus SOD genes were constructed and their SOD activity and resistance to oxidative stresses were measured.

Results

Bioinformatic analysis showed that V. parahaemolyticus SODs were distinguished by their metal cofactors, FeSOD (VP2118), MnSOD (VP2860), and CuZnSOD (VPA1514). VP2118 gene promoter activity was significantly higher than the other SOD genes. In a VP2118 gene deletion mutant, SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. The absence of VP2118 resulted in significantly lowered resistance to ROS generated by hydrogen peroxide, hypoxanthine–xanthine oxidase, or Paraquat. Furthermore, both the N- and C-terminal SOD domains of VP2118 were necessary for ROS resistance.

Conclusion

VP2118 is the primary V. parahaemolyticus SOD and is vital for anti-oxidative stress responses.

General significance

The V. parahaemolyticus FeSOD VP2118 may enhance ROS resistance and could promote its survival in the intestinal tract to facilitate host tissue infection.     

Physiological and subjective responses to low relative humidity

In order to investigate the influence of low relative humidity, we measured saccharin clearance time (SCT), frequency of blinking, heart rate (HR), blood pressure, hydration state of skin, transepidermal water loss (TEWL), recovery sebum level and skin temperature as physiological responses. We asked subjects to judge thermal, dryness and comfort sensations as subjective responses using a rating scale. Sixteen non-smoking healthy male students were selected. The pre-room conditions were maintained at an air temperature (Ta) of 25 degrees C and a relative humidity (RH) of 50%. The test room conditions were adjusted to provide a Ta of 25 degrees C and RH levels of 10%, 30% and 50%.RH had no effect on the activity of the sebaceous gland and on cardiovascular reactions like blood pressure and HR. However, it was obvious that low RH affects SCT, the dryness of the ocular mucosa and the stratum corneum of the skin and causes a decrease in mean skin temperature. Under 30% RH, the eyes and skin become dry, and under 10% RH the nasal mucous membrane becomes dry as well as the eyes and skin, and the mean skin temperature decreases. These findings suggested that to avoid dryness of the eyes and skin, it is necessary to maintain an RH greater than 30%, and to avoid dryness of the nasal mucous membrane, it is necessary to maintain an RH greater than 10%. Subjects felt cold immediately after a change in RH while they had only a slight perception of dryness at the change of humidity.     

Genetic characterization of the natural SigB variants found in clinical isolates of Staphylococcus aureus

The SigB concentrations in clinical isolates of Staphylococcus aureus were measured to examine their correlation with the antibiotic resistance. The SigB concentrations in methicillin-resistant S. aureus (MRSA) were higher than in the control strain, N315, and many of methicillin-susceptible S. aureus (MSSA). Sequencing analyses of the sigB genes revealed that the strains exhibiting the high SigB concentrations have three amino acid substitutions in SigB: I11V, D141N, and Q256K. Further analysis using isogenic mutants demonstrated that D141N (or both D141N and Q256K) is essential to maintain the high SigB concentration. These substitutions affected the UV tolerance, but had no effect on the antibiotic resistance. The SigB activity was affected by these substitutions toward the stationary phase, but not during the transient heat shock response.