Phenylpyruvic acid (PPA) is used as a food and feed additive and has a wide range of applications in the pharmaceutical, chemical and other fields. At present, PPA is mainly produced by chemical synthesis. With the green transformation of the manufacturing industry, biotransformation will be a good alternative for PPA production. The L-amino acid deaminase (PmiLAAD) from Proteus mirabilis has been widely studied for the production of PPA. However, the low yield limits its industrial production. To further enhance the production of PPA and better meet industrial demands, a more efficient synthesis method for PPA was established. In this study, PmiLAAD was heterologously expressed in Escherichia coli. Subsequently, a colorimetric reaction method was established to screen the strains with high PPA production. The semi-rational design of PmiLAAD was carried out, and the obtained triple-site mutant V18 (V437I/S93C/E417A) showed a 35% increase in catalytic activity compared with the wild type. Meanwhile, the effect of N-terminal truncation on the catalytic activity of the V18 mutant was investigated. After the optimization of the whole-cell conditions for the obtained mutant V18-N7, fed-batch conversion was carried out in a 5-L fermenter, and 44.13 g/L of PPA was synthesized with a conversion rate of 88%, which showed certain potential for industrial application. This study lays foundation for the industrial production of phenylpyruvic acid and also offers insights into the biosynthesis of other chemicals.
Escherichia coli/metabolism* ; Proteus mirabilis/genetics* ; Phenylpyruvic Acids/metabolism* ; Protein Engineering/methods* ; Recombinant Proteins/biosynthesis* ; Bacterial Proteins/metabolism*

To create flexible and high-quality pediatric continuing education opportunities, the Children's Hospital of Zhejiang University School of Medicine has developed an online teaching system based on the medical consortium information system, mobile application, and website, which includes "5G+" digital classroom for case teaching, hierarchical diagnosis and treatment tracking, mobile courses, and live interactive lectures about difficult medical cases as support extensions of offline training. From 2020 to 2023, over 9 000 people participated in online activities. The results of questionnaire survey, theoretical examination, and semi-structured interview showed that the members of the medical consortium were satisfied with the training content and effect of the online medical continuing education model; medical personnel's theoretical levels were improved after training; the interviewees believed that online training was flexible and convenient with regard to time, location, and space, with positive impact for improving clinical ability and personal development. However, the online form cannot yet replace the traditional refresher training completely. In the future, we will establish a unified evaluation and assessment mechanism, form a standardized management system, and introduce advanced technologies, so as to encourage enthusiastic and effective participation, and promote the rational and efficient use of medical education resources.

The shortage of pediatric resources and inadequate diagnosis and treatment capacity in primary hospitals are key problems for implementing hierarchic care system of children's diseases. To solve this difficulty, Zhejiang province has constructed a three-stage standardized pattern for pediatric diagnosis and treatment ability training for primary doctors, which integrates training promotion, continuing education and practice output. Training promotion includes the training courses, theoretical examination, and the certification of Zhejiang Pediatric Alliance. Continuing education includes mobile teaching resource library and online regular Q&A. Practical output includes graded diagnosis and treatment of children's diseases and public lectures. From 2018 to 2021, a total of 1 418 trainees participated in 7 training sessions, 327 of whom passed the examination. Finally, 53 participants completed the certification of Zhejiang Pediatric Alliance and were included in the hierarchical diagnosis and treatment system for children in Zhejiang Province, expanding the coverage of hierarchical diagnosis and treatment from 32 institutions in 24 districts to 73 medical institutions in 38 districts. Members of the alliance completed 40 public lectures, with a total audience of 13 233. The three-stage standardized pattern continuously improves the comprehensive ability of primary doctors by means of multi-forms, standardization and continuity. We hope our experience can provide a reference for the training of primary doctors and the construction of hierarchical diagnosis and treatment system in various regions.