Development of a gene-targeting system using CRISPR/Cas9 and utilization of pyrG as a novel selectable marker in Lentinula edodes.

First, we attempted to recombine the Shiitake (Lentinula edodes) pyrG (ura3) gene homologously by introducing a donor vector containing a carboxin resistance gene (lecbxR) flanked by homologous sequences of pyrG into protoplasts of the fungus. However, all the carboxin-resistant transformants only contained ectopic insertions of the exogenous gene and no homologous insertions. Agaricomycetes are generally known for their low efficiency of homologous recombination, and a similar result was shown for L. edodes. We then co-introduced a Cas9 plasmid vector containing a CRISPR/Cas9 expression cassette targeting pyrG and donor plasmid vector. As a result, ∆pyrG strains containing the expected homologous recombination were obtained. However, only two of the seven ∆pyrG strains had the Cas9 sequence; the others did not. Our results suggest that genome editing occurred via the transient expression of the CRISPR/Cas9 cassette in the Cas9 plasmid vector introduced into the fungal cell. Transforming pyrG into a ∆pyrG strain (strain I8) resulted in prototrophic strains with an efficiency of 6.5 strains/experiment.

[Recent trends and challenges for older adults at community sport clubs in Japan: Analysis of FY 2016 Survey Results on Comprehensive Community Sports Clubs].

Objectives　Comprehensive community sports clubs (CCSCs), of which more than 3,500 exist in Japan, have the potential to contribute to healthy aging in older adults, but their effect on this population has not, thus far, been well explored. This study aimed to investigate the current environment affecting seniors at Japanese CCSCs and to examine issues identified by the analysis.Methods　Data were collected from the FY 2016 Survey Results on Comprehensive Community Sports Clubs, conducted by the Japan Sports Agency. A total of 2,444 clubs were divided equally into four groups after calculating the proportion of senior members (ages 60 and over) per total number of members in each club. The groups were then ranked from lowest to highest according to proportion of senior members: Groups A, B, C and D. Additionally, the 2,444 clubs were classified into six regions according to location. Group designation (A to D) and regional classification were used as independent variables. The dependent variables were categorized as follows: basic information (e.g. total number of members), crisis management and accident prevention measures (13 specific items in total), and each club's compliance with legal obligations.Results　Study results revealed that Group D, containing the highest proportion of senior members, had fewer total members, lower monthly membership fees, lower annual club income, less annual club budget apportioned to each member, and fewer instructors than other groups. Regarding crisis management and accident prevention measures, Group D had lower completion rates on 6 items (health certificate submissions, liability insurance enrollment, safety workshop implementation, heatstroke prevention, liaison with healthcare professionals, and AED availability). Group D also showed a lower rate of compliance with legal obligations than other groups. In comparisons between the six regions, the median proportion of senior members was found to be highest in Chugoku-Shikoku and lowest in Chubu, although each median ranged from only 20% to 30%. Regarding crisis management and accident prevention measures, clubs in Kanto region had the highest completion rates for 10 items, whereas clubs in Kinki region had the lowest completion rates for 8 items.Conclusion　The CCSCs with higher proportions of senior members had smaller budgets, fewer members and staff, and delayed implementation of crisis management and accident prevention measures. Regional disparities were also observed both in club scale and management of medical and safety issues. Although our study identified several shortcomings in this area, medical and safety management implementation is an important consideration for CCSCs with high proportions of senior members, as these members are at higher risk for disease and frailty. Affected CCSCs and relevant authorities should therefore acknowledge and address this issue cooperatively.