The sonication-assisted whisker method enables CRISPR-Cas9 ribonucleoprotein delivery to induce genome editing in rice.

CRISPR/Cas9-based genome editing represents an unprecedented potential for plant breeding. Unlike animal cells, plant cells contain a rigid cell wall, genome editing tool delivery into plant cells is thus challenging. In particular, the delivery of the Cas9-gRNA ribonucleoprotein (RNP) into plant cells is desired since the transgene insertion into the genome should be avoided for industrial applications in plants. In this study, we present a novel RNP delivery approach in rice. We applied the sonication-assisted whisker method, conventionally developed for DNA delivery in plants, for RNP delivery in rice. Combined with marker gene delivery, we successfully isolated OsLCYß genome-edited lines generated by RNPs. The calli and regenerated shoot of the OsLCYß mutant showed abnormal carotenoid accumulation. In addition, we also detected, although at a low frequency, genome editing events in rice calli cells by RNP delivery using the sonication-assisted whisker method without any additional. Therefore, the sonication-assisted whisker method could be an attractive way to create RNP-based genome-edited lines in plants.

Risk factors for acute kidney injury in vancomycin and piperacillin/tazobactam combination therapy: A retrospective study.

INTRODUCTION: Combined use of vancomycin (VCM) and piperacillin/tazobactam (PIPC/TAZ) has been reported to increase the incidence of acute kidney injury (AKI). However, the risk factors associated with AKI after VCM and PIPC/TAZ (VPT) administration have not yet been identified. Therefore, we retrospectively assessed patients treated with VPT to investigate the risk factors for AKI development. METHODS: The study involved patients who were treated with VPT from January 1, 2016 to March 31, 2020. The patients were divided into the AKI or non-AKI group. The clinical characteristics of patients and antimicrobial therapy were compared between the groups. Their association with AKI risk was evaluated using multivariate logistic regression analysis. RESULTS: In total, 182 patients were included, with 118 in the non-AKI group and 64 in the AKI group. Therefore, the incidence of AKI was 35.2 %. The initiation of VPT combination therapy on the same day and concomitant use of vasopressors were associated with an increased risk of AKI (odds ratio [OR] 2.55, 95 % confidential interval [CI] 1.20-5.44 and OR 3.22, 95 % CI 1.31-7.89, respectively). CONCLUSION: Our findings suggest that the concomitant use of vasopressors and initiating VPT combination therapy on the same day are likely risk factors for AKI development.

Environmental risk assessment of transgenic miraculin-accumulating tomato in a confined field trial in Japan.

The commercial use of genetically modified (GM) crops requires prior assessment of the risks to the environment when these crops are grown in the field or distributed. Assessments protocols vary across countries and GM crop events, but there is a common need to assess environmental biosafety. In this study, we conducted an environmental risk assessment in a confined field of GM tomato plants that can produce miraculin, a taste-altering protein that causes sour tastes to be perceived as sweet, for practical use in Japan. The evaluation was conducted for 1) competitiveness (the ability to compete with wild plants for nutrients, sunlight, and growing areas and prevent their growth) and 2) the production of toxic substances (the ability to produce substances that interfere with the habitat and growth of wild plants, animals, and microorganisms). Investigations of plant morphology and growth characteristics as well as tolerance to low temperature during early growth and overwintering for assessment endpoints related to competitiveness showed no biologically meaningful difference between GM tomato and non-GM tomato. In addition, harmful substances in plant residues and root secretions were assessed by the plow-in method, succeeding crop test and soil microflora tests, and it was determined that GM tomato does not exhibit an increase in harmful substances. Based on these results, it was concluded that GM miraculin-accumulating tomato is comparable to conventional tomato and is unlikely to have unintended adverse effects in the natural environment of Japan.

Effect of Flooding and the nosZ Gene in Bradyrhizobia on Bradyrhizobial Community Structure in the Soil.

We investigated the effects of the water status (flooded or non-flooded) and presence of the nosZ gene in bradyrhizobia on the bradyrhizobial community structure in a factorial experiment that examined three temperature levels (20°C, 25°C, and 30°C) and two soil types (andosol and gray lowland soil) using microcosm incubations. All microcosms were inoculated with Bradyrhizobium japonicum USDA6T, B. japonicum USDA123, and B. elkanii USDA76T, which do not possess the nosZ gene, and then half received B. diazoefficiens USDA110Twt (wt for the wild-type) and the other half received B. diazoefficiens USDA110ΔnosZ. USDA110Twt possesses the nosZ gene, which encodes N2O reductase; 110ΔnosZ, a mutant variant, does not. Changes in the community structure after 30- and 60-d incubations were investigated by denaturing-gradient gel electrophoresis and an image analysis. USDA6T and 76T strains slightly increased in non-flooded soil regardless of which USDA110T strain was present. In flooded microcosms with the USDA110Twt strain, USDA110Twt became dominant, whereas in microcosms with the USDA110ΔnosZ, a similar change in the community structure occurred to that in non-flooded microcosms. These results suggest that possession of the nosZ gene confers a competitive advantage to B. diazoefficiens USDA110T in flooded soil. We herein demonstrated that the dominance of B. diazoefficiens USDA110Twt within the soil bradyrhizobial population may be enhanced by periods of flooding or waterlogging systems such as paddy-soybean rotations because it appears to have the ability to thrive in moderately anaerobic soil.