L-tyrosine is one of the 20 amino acids that make up proteins and is an essential amino acid for mammals, often used as a nutritional supplement. The conventional methods for synthesizing L-tyrosine have some problems such as the production of many by-products, high requirements for production conditions, and environmental pollution. In this study, we designed and constructed a multi-enzyme cascade for the synthesis of L-tyrosine with alanine, glutamate, ammonium chloride, and phenol as substrates. Initially, the sources of glutamate oxidase, alanine aminotransferase, and tyrosine phenol lyase were screened and analyzed, which was followed by the identification of the rate-limiting enzyme in the reaction process. A colorimetric screening method was established, and the rate-limiting enzyme DbAlaA was engineered to enhance its activity by 40.0%. Subsequently, the reaction conditions, including temperature, pH, cell concentration, and surfactant and coenzyme dosages, were optimized. After optimization, the yield of L-tyrosine reached 9.93 g/L, with a alanine conversion rate of 54.90%. Finally, a feed-batch fermentation strategy was adopted, and the yield of L-tyrosine reached 56.07 g/L after 24 h, with a alanine conversion rate of 65.22%. This study provides a reference for the whole-cell catalytic synthesis of L-tyrosine and its industrialization.
Tyrosine/biosynthesis* ; Escherichia coli/metabolism* ; Tyrosine Phenol-Lyase/genetics* ; Multienzyme Complexes/metabolism* ; Fermentation

Objective:To investigate the efficacy and safety of tirofiban combined with human urinary kallidinogenase (HUK) in the treatment of acute isolated brainstem infarction (AIBI) with early neurological deterioration (END).Methods:Consecutive patients with AIBI complicated with END admitted to Xuzhou Municipal Hospital Affiliated to Xuzhou Medical University within 24 hours of symptom onset from April 2017 to October 2022 were included retrospectively. The patients were divided into a tirofiban group and a combined therapy group. In the tirofiban group, tirofiban was administered via intravenous infusion for 48 hours within 2 hours after END, followed by dual antiplatelet therapy with aspirin and clopidogrel 4 hours before discontinuation of tirofiban. The combined therapy group received HUK treatment in addition to tirofiban. The primary outcome was assessed using the modified Rankin Scale at 3 months after onset, and a score ≤2 was defined as good outcome. The secondary outcome measures included all adverse events during the treatment and follow-up. The clinical data were collected, and univariate and multivariate logistic regression was used to analyze the efficacy and safety of tirofiban combined with HUK. Results:A total of 256 patients with AIBI complicated with END were included. The median age was 64 (interquartile range, 56-71) years, and 185 patients were males (72.27%). One hundred and eighteen patients (46.09%) received combined treatment, and 138 (53.91%) received tirofiban alone; 165 (64.45%) had good outcome, and 91 (35.55%) had poor outcome. There was no significant difference in demographic baseline data between the combined therapy group and the tirofiban group. The rate of good outcome at 90 days after onset in the combined treatment group was significantly higher than that in the tirofiban group (72.03% vs. 57.97%; χ 2=5.491, P=0.019), but there were no significant differences in the incidence of symptomatic intracranial hemorrhage, asymptomatic hemorrhagic transformation, mortality within 90 days, and serious adverse events. Multivariate logistic regression analysis showed that the combined therapy was an independent protective factor for good outcome (odds ratio 0.388, 95% confidence interval 0.224-0.672; P<0.001), while diabetes was an independent risk factor for poor outcome (odds ratio 1.530, 95% confidence interval 1.313-1.784; P<0.001). Conclusion:Early combined use of tirofiban and HUK therapy can effectively improve the outcome of patients with AIBI complicated with END, and has good safety.

Objective:To evaluate the effectiveness of entrustable professional activities (EPAs) in the general practice internship of undergraduate clinical medicine students, identify issues that need improvement in the internship, and enhance medical students' competence.Methods:A total of 75 students in the five-year (English class) clinical medicine program enrolled in 2018 and 2019 who participated in general practice internship in Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine from October 2021 to October 2023 were selected as study subjects. The design of core EPAs was adopted to assess the correlation among different EPA dimensions and to analyze the qualified rates.Results:The evaluation of EPAs showed that EPA2 (practicing respect, understanding and teamwork) had the highest mean score of 9.33, and EPA10 (chronic disease management and management of key populations) had the lowest mean score of 8.08. A supervision level of 3a and above was used as the criterion for qualification. The supervision levels of the students' EPAs were mostly concentrated at levels 3a and 3b. The highest qualified rate was for EPA2 (practicing respect, understanding and teamwork) at 85.33%, followed by EPA1 (complying with the rules of the profession and demonstrating professionalism) at 80.00% and EPA8 (reviewing information and solving clinical problems) at 72.00%. The lowest qualified rate was for EPA10 (chronic disease management and management of key populations) at 33.33%, followed by EPA4 (analyzing and interpreting test results) at 57.33%.Conclusions:EPAs concretize competency evaluation, which can effectively reflect the "competency-oriented" training objectives encompassing multiple elements such as knowledge, skills, values, and attitudes, while maintaining professional specificity. Undergraduates demonstrated strengths in professionalism and academics, but showed deficiencies in community chronic disease management and management of key populations. These findings suggest the need to strengthen the training in health and social care to better align with the competencies required during standardized residency training.

Objective:To evaluate the effectiveness of entrustable professional activities (EPAs) in the general practice internship of undergraduate clinical medicine students, identify issues that need improvement in the internship, and enhance medical students' competence.Methods:A total of 75 students in the five-year (English class) clinical medicine program enrolled in 2018 and 2019 who participated in general practice internship in Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine from October 2021 to October 2023 were selected as study subjects. The design of core EPAs was adopted to assess the correlation among different EPA dimensions and to analyze the qualified rates.Results:The evaluation of EPAs showed that EPA2 (practicing respect, understanding and teamwork) had the highest mean score of 9.33, and EPA10 (chronic disease management and management of key populations) had the lowest mean score of 8.08. A supervision level of 3a and above was used as the criterion for qualification. The supervision levels of the students' EPAs were mostly concentrated at levels 3a and 3b. The highest qualified rate was for EPA2 (practicing respect, understanding and teamwork) at 85.33%, followed by EPA1 (complying with the rules of the profession and demonstrating professionalism) at 80.00% and EPA8 (reviewing information and solving clinical problems) at 72.00%. The lowest qualified rate was for EPA10 (chronic disease management and management of key populations) at 33.33%, followed by EPA4 (analyzing and interpreting test results) at 57.33%.Conclusions:EPAs concretize competency evaluation, which can effectively reflect the "competency-oriented" training objectives encompassing multiple elements such as knowledge, skills, values, and attitudes, while maintaining professional specificity. Undergraduates demonstrated strengths in professionalism and academics, but showed deficiencies in community chronic disease management and management of key populations. These findings suggest the need to strengthen the training in health and social care to better align with the competencies required during standardized residency training.

Objective:To investigate the predictive factors of early neurological deterioration (END) in patients with single subcortical infarction (SSI).Methods:Consecutive patients with SSI within 12 h of onset admitted to the Department of Neurology, Xuzhou Municipal Hospital Affiliated to Xuzhou Medical University from January 2016 to December 2021 were included retrospectively. The clinical and imaging data of the END and non-END groups were collected and compared. Multivariate logistic regression analysis was used to determine the independent risk factors for END. Results:A total of 1 060 patients with SSI were included, of which 268 (25.28%) developed END. There were statistically significant differences in the infarct involving internal capsule on diffusion-weighted imaging, SSI classification, maximum diameter and classification of the infarcts, and the number of infarct layers between the END group and the non-END group (all P<0.05). Multivariate logistic regression analysis showed that the infarct involved the internal capsule (odds ratio [ OR] 1.52, 95% confidence interval [ CI] 1.27-1.96; P=0.015), the maximum diameter of the infarct ( OR 1.33, 95% CI 1.12-1.42; P=0.023), proximal SSI ( OR 1.49, 95% CI 1.28-1.92; P=0.018), and the number of infarct layers (compared to 1 layer, 3 layers: OR 15.01, 95% CI 4.12-12.45, P=0.013; ≥ 4 layers: OR 15.42, 95% CI 5.67-18.43, P=0.004) were independent risk factors for END. Conclusion:Infarct involved internal capsule on diffusion-weighted imaging, larger diameter of the infarct, proximal SSI, and ≥3 layers of infarct may be predictive factors of the occurrence of END in patients with SSI.

Objective:This study aimed to evaluate the efficacy of decitabine (DAC) and identify factors influencing treatment responses in patients with primary immune thrombocytopenia (ITP) who had failed glucocorticoid therapy.Methods:Clinical data of 61 patients with glucocorticoid-resistant ITP who received DAC therapy (5 mg·m -2·d -1×3 d via intravenous infusion) for at least three cycles with 3-4-week intervals at the Department of Hematology, Qilu Hospital of Shandong University, from November 2015 to June 2021 were analyzed retrospectively. Results:The 61 patients comprised 20 males and 41 females, with a median age of 45 years (range: 15-81 years). Among them, 43 patients were glucocorticoid-dependent (glucocorticoid-dependent group), while 18 patients were glucocorticoid-resistant (glucocorticoid-resistant group). Following DAC treatment, 12 patients (19.67% ) achieved complete response (CR), and 16 patients (26.23% ) exhibited response (R), resulting in an overall response (OR) rate of 45.90% (28/61). Comparison between the OR group ( n=28) and the non-response (NR) group ( n=33) revealed significant differences in responses to glucocorticoids (dependent or resistant) and platelet counts before treatment ( χ2=8.789, P=0.003; z=-2.416, P=0.016). The glucocorticoid-dependent group showed higher platelet counts than the glucocorticoid-resistant group after the second and third cycles of DAC treatment ( P=0.032, 0.024). Moreover, the OR rates after the first, second, and third cycles of DAC treatment in the glucocorticoid-dependent group were all higher than those in the glucocorticoid-resistant group ( P=0.042, P=0.012, P=0.029). A significant correlation was observed between glucocorticoid dependence and responses to DAC treatment ( OR=9.213, 95% CI 1.937-43.820, P=0.005) . Conclusion:DAC demonstrates definitive efficacy with mild adverse effects in a subset of patients with glucocorticoid-resistant primary ITP. Glucocorticoid dependence and higher platelet counts before treatment are associated with a favorable response to DAC therapy.

Objective:To investigate the efficacy and safety of tirofiban in patients with acute ischemic stroke (AIS) with early neurological deterioration (END).Methods:Consecutive patients with AIS who had END and did not receive reperfusion treatment admitted to Xuzhou Municipal Hospital Affiliated to Xuzhou Medical University within 24 h of onset from January 2017 to December 2022 were retrospectively included. END was defined as an increase of ≥2 in the National Institutes of Health Stroke Scale score within 72 h after onset compared at admission, or an increase of 1 in motor function. The tirofiban group was given tirofiban within 2 h after the occurrence of END, while the control group was given aspirin+clopidogrel. A score of ≤2 on the modified Rankin Scale at 90 d after onset was considered as a good outcome.Results:A total of 502 patients with AIS who had END were enrolled; including 322 males (64.14%) aged 65 ± 9 years. There was no statistically significant difference in baseline characteristics between the tirofiban group ( n=252) and the control group ( n=250). The good outcome rate of the tirofiban group at 90 d was significantly higher than that of the control group (60.32% vs. 42.00%; P<0.05), while there were no statistically significant differences in the incidence of symptomatic hemorrhagic transformation, asymptomatic hemorrhagic transformation, death and serious adverse events within 90 d. There were statistically significant differences in diabetes, atrial fibrillation, use of tirofiban, and the classification of stroke etiology between the good outcome group and the poor outcome group ( P<0.05). Multivariate logistic regression analysis showed that the use of tirofiban was an independent correlated factor for good outcome (odds ratio 0.33, 95% confidence interval 0.22-0.48; P<0.001). Conclusion:Tirofiban has good efficacy and safety for END in patients with AIS who did not received reperfusion therapy.

As one of the first institutions to carry out integrated education in China, the Shanghai Jiao Tong University School of Medicine has carried out a pilot reform of the organ system centered vertical single cycle integrated curriculum in the undergraduate medical education (five-year English stream) based on the Sino-foreign cooperative education program. The curriculum has been formed with multi-disciplinary single cycle integrated courses, longitudinal integrated courses, and three-dimensional specialized expansion electives. The curriculum focuses on the competency training of medical student. The aim is to highlight the teaching concept of early contact with clinic, attach importance to the cultivation and shaping of medical personality, achieve effective connection with post-graduation education, and realize the intersection and deep integration of social, humanities and medical professional courses through the construction of a series of course modules.

Objective:To investigate the correlation between fibrinogen and lipoprotein (a) and early neurological deterioration (END) in acute ischemic stroke patients with diabetes.Methods:From January 2017 to December 2020, patients with acute ischemic stroke admitted to the Department of Neurology, Xuzhou Municipal Hospital Affiliated to Xuzhou Medical University within 48 h of onset were enrolled retrospectively. END was defined as the National Institutes of Health Stroke Scale (NIHSS) score within 7 d after onset increased by ≥2 or motor function score increased by ≥1 compared with the baseline. Demographic and baseline clinical data were collected. Multivariate logistic regression analysis was used to identify the independent risk factors for END in general and diabetic patients with acute ischemic stroke. Results:A total of 1 504 patients with acute ischemic stroke were enrolled. Two hundred and fifty-two (16.76%) patients had END. The age, baseline NIHSS score, random blood glucose, fibrinogen, lipoprotein (a) levels, and the proportion of patients with diabetes in the END group were higher than those in the non-END group. There were also significant differences in various stroke etiologic subtypes between the END group and the non-END group (all P<0.05). Multivariate logistic regression analysis showed that fibrinogen and lipoprotein (a) levels were not the independent risk factors for END in patients with acute ischemic stroke. Three hundred and thirty-seven patients also had diabetes mellitus, of which 85 had END (25.22%). The levels of fibrinogen and lipoprotein (a) in the END group were significantly higher than those in the non-END group ( P<0.05). Multivariate logistic regression analysis showed that fibrinogen (odds ratio 2.23, 95% confidence interval 1.75-4.54; P=0.002) and lipoprotein (a) (odds ratio 1.98, 95% confidence interval 1.57-3.65; P=0.003) were the independent risk factors for END in acute ischemic stroke patients with diabetes. Conclusion:Higher fibrinogen and lipoprotein (a) levels are associated with END in acute ischemic stroke patients with diabetes.

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.
Asparaginase/genetics* ; Bacillus subtilis/genetics* ; Industrial Microbiology ; Protein Engineering ; Rhizomucor/enzymology* ; Sequence Alignment