CRISPR/Cas9-Based Genome Editing for Protein Expression and Secretion in Kluyveromyces lactis.

The budding yeast Kluyveromyces lactis has emerged as a promising microbial chassis in industrial biotechnology. However, a lack of efficient molecular genetic manipulation tools and strategies has hindered the development of K. lactis as a biomanufacturing platform. In this study, we developed and applied a CRISPR/Cas9-based genome editing method to K. lactis. Single-gene editing efficiency was increased to 80% by disrupting the nonhomologous end-joining-related gene KU80 and performing a series of process optimizations. Subsequently, the CRISPR/Cas9 system was explored based on different sgRNA delivery modes for simultaneous multigene editing. With the aid of the color indicator, the editing efficiencies of two and three genes reached 73.3 and 36%, respectively, in the KlΔKU80 strain. Furthermore, the CRISPR/Cas9 system was used for multisite integration to enhance lactase production and combinatorial knockout of TMED10 and HSP90 to characterize the extracellular secretion of lactase in K. lactis. Generally, genome editing is a powerful tool for constructing K. lactis cell factories for protein and chemical production.

Bedtime procrastination related to loneliness among Chinese university students during post-pandemic period: a moderated chain mediation model.

BACKGROUND: This study examined the relationship between loneliness and bedtime procrastination among Chinese university students, the mediating effects of COVID-19 risk perception and self-regulatory fatigue, and connectedness to nature's protective role, post pandemic. METHODS: We recruited 855 students to complete the Loneliness, Perceived Risk of COVID-19 Pandemic, Self-Regulatory Fatigue, Bedtime Procrastination, and Connectedness to Nature Scales. Data for descriptive statistics, correlation analysis, and moderated chain mediation effects were analyzed using SPSS 24.0 and process 3.5 macros. RESULTS: Loneliness positively correlated with bedtime procrastination, COVID-19 risk perception mediated the impact of loneliness on bedtime procrastination, self-regulatory fatigue mediated the effect of loneliness on bedtime procrastination, and COVID-19 risk perception and self-regulatory fatigue mediated the effect between loneliness and bedtime procrastination. Furthermore, connectedness to nature mediated the impact of COVID-19 risk perception on self-regulatory fatigue. CONCLUSIONS: The results indicate the effects and potential mechanisms of loneliness on bedtime procrastination after the relaxation of the pandemic prevention and control policy in China from the perspective of self-regulatory resources and provide insights into improving university students' sleep routine and mental health post pandemic.

Oxygenation alleviates waterlogging-caused damages to cherry rootstocks.

Waterlogging has occurred more frequently in recent years due to climate change, so it is a huge threat to crop yield and quality. Sweet cherry, a fruit tree with a high economic value, is sensitive to waterlogging stress. One of the most effective methods for enhancing the waterlogging tolerance of sweet cherries is to select waterlogging-tolerant rootstocks. However, the waterlogging tolerance of different cherry rootstocks, and the underlying mechanism remains uncharacterized. Thus, we first evaluated the waterlogging resistance of five sweet cherry rootstocks planted in China. The data showed that 'Gisela 12' and 'Colt' were the most waterlogging-sensitive and -tolerant among the five tested varieties, respectively. Oxygenation effectively alleviated the adverse impacts of waterlogging stress on cherry rootstocks. Moreover, we found that the waterlogging group had lower relative water content, Fv/Fm value, net photosynthetic rate, and higher antioxidant enzyme activities, whereas the oxygenated group performed better in all these parameters. RNA-Seq analysis revealed that numerous DEGs were involved in energy production, antioxidant metabolism, hormone metabolism pathways, and stress-related transcription factors. These findings will help provide management strategies to enhance the waterlogging tolerance of cherry rootstocks and thereby achieve higher yield and better quality of cherries.

Research on the application and effect of flipped-classroom combined with TBL teaching model in WeChat-platform-based biochemical teaching under the trend of COVID-19.

BACKGROUND: Biochemistry is a core subject in clinical medical education. The traditional classroom teaching model led by teachers is often limited to the knowledge transfer of teachers and the passive acceptance of students. It lacks interactive and efficient teaching methods and is not enough to meet the learning needs and educational goals of modern students. The combination of WeChat public platform, flipped classroom and TBL teaching model is closer to the needs of real life and workplace, helping students to cultivate comprehensive literacy and the ability to solve practical problems. At the same time, this teaching model has yet to be used in biochemistry courses. OBJECTIVE: To explore the influence of the mixed teaching model of flipped classroom and combining TBL based on WeChat public platform upon undergraduates in biochemistry. METHODS: Using the mixed research method of quasi-experimental research design and descriptive qualitative research, 68 students were selected into the traditional and the blended teaching groups. Among them, the blended teaching group adopts the blended teaching model of flipped classroom combined with TBL based on the WeChat platform to learn biochemical courses. In this study, an independent sample t-test was intended to analyze the differences in final scores, a chi-square test was served to analyze the differences in satisfaction questionnaires, and thematic analysis was used to analyze semi-structured interview data. RESULTS: Compared with the traditional teaching model, the mixed teaching model significantly improved students' final exam scores (P < 0.05). The teaching satisfaction of the mixed teaching group was also higher than that of the traditional teaching group with statistical significance (P < 0.05). The results of the interviews with eight students were summarized into three topics: (1) Stimulating interest in learning; (2) Improving the ability of autonomous learning; (3) Recommendations for improvement. CONCLUSIONS: The combination of the WeChat platform and flipped classroom with TBL has a positive effect on improving medical students' autonomous learning ability and problem-solving ability. The research shows that the teaching mode of flipped classroom combined with TBL based on the WeChat platform is effective and feasible.

A Systematic Review of Plantar Plate Repair in the Management of Lesser Metatarsophalangeal Joint Instability.

The plantar plate is a major structure that maintains metatarsophalangeal joint (MTPJ) stability and has only recently gained attention. Anatomic plantar plate repair can directly address the pathologic entity, rather than relying on indirect reduction of the MTPJ instability by osteotomy or tendon transfer techniques. The present report aimed to determine the effectiveness of plantar plate repair for the treatment of patients with lesser MTPJ instability. Different databases were searched using the guidelines in the Cochrane Handbook and recommendations from the Preferred Reporting Items for Systematic reviews and Meta-Analysis statement. Six case series, describing 162 patients who had undergone plantar plate repair in conjunction with either Weil osteotomy or flexor digitorum longus transfer, were retrieved. Favorable outcomes were described in each of the studies included in the present systematic review. However, these results should be interpreted with caution because of the methodologic limitations and biases inherent in the included studies. More rigorous clinical investigations are required to fully understand the effectiveness of plantar plate repair for the management of lesser MTPJ instability.