Efficient genome editing with CRISPR/Cas9 in Pleurotus ostreatus

Pleurotus ostreatus is one of the most commercially produced edible mushrooms worldwide. Improved cultivated strains with more useful traits have been obtained using classical breeding, which is laborious and time-consuming. Here, we attempted efficient gene mutagenesis using plasmid-based CRISPR/Cas9 as the first step for non-genetically modified (non-GM) P. ostreatus generation. Plasmids harboring expression cassettes of Cas9 and different single guide RNAs targeting fcy1 and pyrG were individually transferred into fungal protoplasts of the PC9 strain, which generated some strains exhibiting resistance to 5-fluorocytosine and 5-fluoroorotic acid, respectively. Genomic PCR followed by sequencing revealed small insertions/deletions or insertion of a fragment from the plasmid at the target site in some of the drug-resistant strains. The results demonstrated efficient CRISPR/Cas9-assisted genome editing in P. ostreatus, which could contribute to the molecular breeding of non-GM cultivated strains in the future. Furthermore, a mutation in fcy1 via homology-directed repair using this CRISPR/Cas9 system was also efficiently introduced, which could be applied not only for precise gene disruption, but also for insertions leading to heterologous gene expression in this fungus

Functional and expressional analyses of apple FLC-like in relation to dormancy progress and flower bud development.

We previously identified the FLOWERING LOCUS C (FLC)-like gene, a MADS-box transcription factor gene that belongs to Arabidopsis thaliana L. FLC clade, in apple (Malus ×domestica Borkh.), and its expression in dormant flower buds is positively correlated with cumulative cold exposure. To elucidate the role of the MdFLC-like in the dormancy process and flower development, we first characterized the phenotypes of MdFLC-like overexpressing lines with the Arabidopsis Columbia-0 background. The overexpression of MdFLC-like significantly delayed the bolting date and reduced the plant size, but it did not significantly affect the number of rosette leaves or flower organ formation. Thus, MdFLC-like may affect vegetative growth and development rather than flowering when expressed in Arabidopsis, which is not like Arabidopsis FLC that affects development of flowering. We compared seasonal expression patterns of MdFLC-like in low-chill ‘Anna’ and high-chill ‘Fuji’ and ‘Tsugaru’ apples collected from trees grown in a cold winter region in temperate zone and found an earlier upregulation in ‘Anna’ compared with ‘Fuji’ and ‘Tsugaru’. Expression patterns were also compared in relation to developmental changes in the flower primordia during the chilling accumulation period. Overall, MdFLC-like was progressively upregulated during flower primordia differentiation and development in autumn to early winter and reached a maximum expression level at around the same time as the genotype-dependent chilling requirements were fulfilled in high-chill cultivars. Thus, we hypothesize MdFLC-like may be upregulated in response to cold exposure and flower primordia development during the progress of endodormancy. Our study also suggests MdFLC-like may have a growth-inhibiting function during the end of endodormancy and ecodormancy when the temperature is low and unfavorable for rapid bud outgrowth

Efficient genome editing with CRISPR/Cas9 in Pleurotus ostreatus

Pleurotus ostreatus is one of the most commercially produced edible mushrooms worldwide. Improved cultivated strains with more useful traits have been obtained using classical breeding, which is laborious and time-consuming. Here, we attempted efficient gene mutagenesis using plasmid-based CRISPR/Cas9 as the first step for non-genetically modified (non-GM) P. ostreatus generation. Plasmids harboring expression cassettes of Cas9 and different single guide RNAs targeting fcy1 and pyrG were individually transferred into fungal protoplasts of the PC9 strain, which generated some strains exhibiting resistance to 5-fluorocytosine and 5-fluoroorotic acid, respectively. Genomic PCR followed by sequencing revealed small insertions/deletions or insertion of a fragment from the plasmid at the target site in some of the drug-resistant strains. The results demonstrated efficient CRISPR/Cas9-assisted genome editing in P. ostreatus, which could contribute to the molecular breeding of non-GM cultivated strains in the future. Furthermore, a mutation in fcy1 via homology-directed repair using this CRISPR/Cas9 system was also efficiently introduced, which could be applied not only for precise gene disruption, but also for insertions leading to heterologous gene expression in this fungus

Temporal increase in muscle cross-sectional area as an acute effect of resistance exercise in resistance-trained and untrained individuals

The purpose of this study was to compare the temporal increase in muscle cross-sectional area (CSA) as the acute response of resistance exercise (RE) between resistance-trained and untrained groups, and investigate the factors that affect the muscle CSA. Resistance-trained (n = 14) and untrained (n = 14) subjects performed four kinds of triceps brachii RE. Muscle CSA and intracellular hydration (IH), were measured prior to and 5-, 30-, and 60-minute after RE. Pearson's correlation coefficient was calculated to clarify the relationships among percent increases in muscle CSA and IH, area under the Oyx-Hb curve, blood lactate concentration, and % maximum voluntary contraction (MVC)-root-mean-square (RMS) of electromyogram (EMG). At 5-minute after RE, muscle CSA increased significantly to 120.2 ± 6.3% in the resistance-trained group and 105.5 ± 2.3% in the untrained group (p < 0.01). However, neither group showed a significant difference between the values before and 30-minute after RE. In the resistance-trained group, there was a significant increase in IH at 5-minute post-RH (p < 0.01), and correlations were found between percent increases in muscle CSA and IH (r = 0.70, p < 0.01), area under the Oxy-Hb curve (r = 0.77, p < 0.01), and % MVC-RMS of EMG (r = 0.72, p < 0.01). The findings of this study suggest that measurements of muscle CSA in studies of muscle hypertrophy should be performed 30-minute or more after the last resistance exercise session, and muscle pump exercises should be conducted just before participation in bodybuilding, and physique contests

Association of L-type amino acid transporter 1 (LAT1) with the immune system and prognosis in invasive breast cancer

L-type amino acid transporter 1 (LAT1), also referred to as SLC7A5, is believed to regulate tumor metabolism and be associated with tumor proliferation. In invasive breast cancer, we clinicopathologically investigated the utility of LAT1 expression. LAT1 expression was evaluated via immunohistochemistry analyses in 250 breast cancer patients undergoing long-term follow-up. We assessed the relationships between LAT1 expression and patient outcomes and clinicopathological factors. Breast cancer-specific survival stratified by LAT1 expression was assessed. Human epidermal growth factor receptor 2 (HER2)-positive patients with metastasis received trastuzumab therapy. The density of tumor-infiltrating lymphocytes (TILs) was evaluated according to the International Working Group guidelines. In the current study, high LAT1 expression was significantly correlated with estrogen receptor (ER) negativity, progesterone receptor negativity, high histological grade, increased TILs, and programmed death ligand 1 positivity. Among the ER-positive and HER2-negative patients, high LAT1 was an independent indicator of poor outcomes (hazard ratio (HR) = 2.97; 95% confidence interval (CI), 1.16–7.62; p = 0.023). Moreover, high LAT1 expression was an independent poor prognostic factor in luminal B-like breast cancer with aggressive features (HR = 3.39; 95% CI 1.35–8.52; p = 0.0094). In conclusion, high LAT1 expression could be used to identify a subgroup of invasive breast cancer characterized by aggressive behavior and high tumor immunoreaction. Our findings suggest that LAT1 might be a candidate therapeutic target for breast cancer patients, particularly those with luminal B-like type breast cancer

The effect of Humanitude care methodology on improving empathy: a six-year longitudinal study of medical students in Japan.

BACKGROUND: Empathy, which involves understanding another person\u27s experiences and concerns, is an important component for developing physicians\u27 overall competence. This longitudinal study was designed to test the hypothesis that medical students\u27 empathy can be enhanced and sustained by Humanitude Care Methodology, which focuses on perception, emotion and speech. METHODS: This six-year longitudinal observational study examined 115 students who entered Okayama University Medical School in 2013. The study participants were exposed to two empathy-enhancing programs: (1) a communication skills training program (involving medical interviews) and (2) a Humanitude training program aimed at enhancing their empathy. They completed the Jefferson Scale of Empathy (JSE) seven times: when they entered medical school, before participation in the first program (medical interview), immediately after the first program, before the second program (Humanitude exercise), immediately after the second program, and in the 5th and 6th year (last year) of medical school. A total of 79 students (69% of the cohort) completed all seven test administrations of the JSE. RESULTS: The mean JSE scores improved significantly after participation in the medical interview program (p \u3c 0.01) and the Humanitude training program (p = 0.001). However, neither program showed a sustained effect. CONCLUSIONS: The Humanitude training program as well as medical interview training program, had significant short-term positive effects for improving empathy among medical students. Additional reinforcements may be necessary for a long-term sustained effect

The Stellar Abundances for Galactic Archaeology (SAGA) Database II - Implications for Mixing and Nucleosynthesis in Extremely Metal-Poor Stars and Chemical Enrichment of the Galaxy

We discuss the characteristics of known extremely metal-poor (EMP) stars in the Galaxy using the Stellar Abundances for Galactic Archaeology (SAGA) database (Suda et al. 2008, PASJ, 60, 1159).The analyses of carbon-enhanced stars in our sample suggest that the nucleosynthesis in AGB stars can contribute to the carbon enrichment in a different way depending on whether the metallicity is above or below [Fe/H] ~ -2.5, which is consistent with the current models of stellar evolution at low metallicity. We find the transition of the initial mass function at [Fe/H] ~ -2 in the viewpoint of the distribution of carbon abundance and the frequency of carbon-enhanced stars. For observed EMP stars, we confirmed that some, not all, of observed stars might have undergone at least two types of extra mixing to change their surface abundances. One is to deplete the lithium abundance during the early phase of red giant branch. Another is to decrease the C/N ratio by one order of magnitude during the red giant branch phase. Observed small scatters of abundances for alpha-elements and iron-group elements suggest that the chemical enrichment of our Galaxy takes place in a well-mixed interstellar medium. We find that the abundance trends of alpha-elements are highly correlated with each other, while the abundances of iron-group elements are subject to different slopes relative to the iron abundance. This implies that the supernova yields of alpha-elements are almost independent of mass and metallicity, while those of iron-group elements have a metallicity dependence or mass dependence with the variable initial mass function.The occurrence of the hot bottom burning in the mass range of 5 <~ M / Msun <~ 6 is consistent with the initial mass function of the Galaxy peaked at ~ 10 - 12 Msun to be compatible with the statistics of carbon enhanced stars with and without s-process element (truncated)Comment: 35 pages, 27 figures, 6 tables, accepted by MNRAS, database to reproduce figures is available at http://saga.sci.hokudai.ac.j

Технологічні рішення і технічні засоби підвищення екологічної безпеки проведення бурових робіт

Рассмотрено влияние проведения буровых работ на окружающую среду. Приведены рекомендации для повышения экологической безопасности проведения буровых работ. Описаны технологические решения и технические средства уменьшения неблагоприятного влияния бурения нефтегазовых скважин на окружающую среду. В комплексе решены и эффективно внедрены на практике вопросы экологической безопасности проведения буровых работ, утилизации, размещения и захоронения отходов бурения. Дказано, что безамбарное бурение на месторождениях возможно при наличии шламонакопителя и нагнетательной скважины.The environmental impacts of drilling works are considered. A range of recommendations for environmental safety enhancement of drilling works is given. Technological solutions and technical means to reduce adverse environmental impacts of oil and gas wells drilling are described. The issues of drilling works environmental safety, utilization, disposal and burial of waste are jointly solved and successfully implemented. The pit less drilling is proved to be possible for any field under the condition of slurry pond and injection well use