L-theanine is an important natural non-protein amino acid that is widely used in food and medicine. Although in previous studies, a microbial fermentation method for L-theanine without the addition of ethylamine has been developed, the conversion rate of this process needs to be further improved. In this study, we constructed a de novo synthesis pathway of L-theanine with glucose as the substrate. First, an in vitro transformation pathway containing ω-transaminase (TA) and γ-glutamylmethylamide synthetase (GMAS) was designed, optimized, and introduced into the chassis strain Escherichia coli K12 W3110 to achieve de novo synthesis of L-theanine. To improve the synthesis efficiency through metabolic engineering, we increased the copies of the GMAS gene gams and the TA gene spuC and enhanced the expression of the aldehyde dehydrogenase gene eutE to provide sufficient acetaldehyde substrate, knocked out the lactate dehydrogenase gene ldhA and the pyruvate formate lyase gene pflB to block bypass metabolism, and introduced the alanine dehydrogenase gene alD to recycle alanine. Furthermore, we over-expressed the phosphoenolpyruvate carboxylase gene ppc to enhance the carbon flux of the TCA cycle, knocked out the succinyl-CoA synthase gene sucCD to reduce the loss of downstream flux of TCA, and integrated the glutamate dehydrogenase gene gdh to enhance the supply of L-glutamate. Finally, the polyphosphate kinase gene ppk was introduced to the ATP cycle, which enhanced the energy supply in L-theanine production. The recombinant strain Tea11 produced 22.60 g/L L-theanine in a 5 L fermenter in 28 h, with a conversion rate of 41.71%. This synthetic pathway in this study balanced the relationship between the supply of ethylamine and the production of theanine, providing a new idea for metabolic engineering of microorganisms to produce L-theanine.
Glutamates/biosynthesis* ; Metabolic Engineering/methods* ; Escherichia coli/genetics* ; Fermentation ; Transaminases/metabolism* ; Amide Synthases/metabolism* ; Glucose/metabolism*

Humans ; Transcriptome/genetics* ; East Asian People ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; Pulmonary Disease, Chronic Obstructive/genetics* ; Polymorphism, Single Nucleotide/genetics*

Animals ; Rats ; Neuroprotective Agents/pharmacology* ; Amyloid beta-Peptides ; PC12 Cells ; Polyunsaturated Alkamides/pharmacology* ; Apoptosis ; Cell Survival ; Peptide Fragments

Objective:To investigate blood glucose variability, National Institutes of Health Stroke Scale (NIHSS) score, Acute Physiology and Chronic Health Evaluation II (APACHE II) score and prognosis in patients with acute and severe cerebrovascular disease, providing a reference for clinical treatment of this disease.Methods:The clinical data of 76 patients with acute and severe cerebrovascular disease who received treatment in Xixi Hospital of Hangzhou between January 2014 and December 2019 were retrospectively analyzed. These patients were divided into a survival group ( n = 51) and a death group ( n = 25) according to the prognosis 28 days after admission. Baseline data, baseline NIHSS score, APACHE II score, standard deviation of blood glucose, mean blood glucose, mean amplitude of glycemic excursions, and blood glucose variability rate were compared between the survival and death groups. The effects of these parameters on the prognosis of acute and severe cerebrovascular disease were analyzed. Results:There were no significant differences in gender, age, history of diabetes, type of disease, and length of hospital stay between the two groups ( χ2 = 1.674, t = 1.048, χ2 = 3.833, 0.263, t = 0.832, all P > 0.05). The proportion of patients with a history of hypertension in the death group was significantly higher than that in the survival group [15.69% (8/51) vs. 52.00% (13/25), χ2 = 11.063, P < 0.05]. There was no significant difference in mean blood glucose between the two groups ( t = 0.118, P > 0.05). The baseline NIHSS score, APACHE II score, standard deviation of blood glucose, mean amplitude of glycemic excursions and blood glucose variability rate in the death group were (24.41 ± 4.14) points, (25.00 ± 6.97) points, (2.72 ± 0.91) mmol/L, (6.27 ± 2.01) mmol/L, (34.83 ± 5.61) %,which were significantly higher than those in the survival group [(17.22 ± 3.63) points, (19.21 ± 5.36) points, (1.69 ± 0.70) mmol/L, (3.72 ± 1.68) mmol/L, (19.54 ± 3.22) %, t = 7.744, 3.999, 5.448, 5.823, 15.095, all P < 0.05]. The proportion of patients with blood glucose variability rate < 20% in the death group was significantly lower than that in the survival group [16.00% (4/25) vs. 74.51% (38/51), χ2= 23.230, P < 0.05]. The proportion of patients with blood glucose variability rate > 30% in the death group was significantly higher than that in the survival group [60.00% (15/25) vs. 13.73% (7/51), χ2 = 17.466, P < 0.05). Logistic regression analysis revealed that baseline NIHSS score, APACHE II score, standard deviation of blood glucose, mean amplitude of glycemic excursions, and blood glucose variability rate were the independent risk factors of death of cerebrovascular disease patients (all P < 0.05). Conclusion:In patients with acute and severe cerebrovascular disease, an obvious blood glucose fluctuation, high baseline NIHSS score, and high APACHE II score help to assess the prognosis of acute and severe cerebrovascular disease and determine the risk of death.

Objective To analyze the therapeutic effect of emergency surgery in the treatment of severe valvular heart disease and its influence on heart function.Methods 87 cases with severe valvular heart disease were selected,and they were given emergency surgical treatment.Before and after treatment,the cardiac function index[end -diastolic volume (EDV),end-systolic volume (ESV),left ventricular end systolic volume index (LVESVI),left ventricular end-diastolic volume index (LVEDVI),left ventricular ejection fraction (LVEF)],postoperative complications and survival rate were compared.Results After treatment,the EDV [(125.78 ± 31.67) mL],ESV [(71.30 ± 16.58) mL] and LVESVI [(50.39 ± 13.30) mL/m2],LVEDVI [(88.74 ± 13.51) mL/m2],LVEF [(51.48 ± 12.73) ％] were better than those before treatment [(116.48 ± 28.94) mL,(63.26 ± 15.47) mL,(33.27 ± 8.54) mL/m2,(64.47 ± 12.69)mL/m2,(47.53 ± 11.20)％],the differences were statistically significant (t =2.022,2.022,3.307,12.213,2.173,all P ＜ 0.05).The incidence rate of complications was 24.14％ (21/87),included arrhythmia in 4 cases,low cardiac output syndrome in 8 cases,acute renal failure in 9 cases.Postoperative 1 year,2 years,the survival rates were 91.95％ (80/87),85.06％ (74/87),respectively.Conclusion Emergency surgical treatment for severe valvular heart disease can effectively improve cardiac function index,improve the survival rate.

Objective To observe the degree of joint flexion and extension of elbow and knee joints of moderate skeletal fluorosis patients before and after treatment and to assess the effects of Gun's MaQian decoction in treating moderate skeletal fluorosis. Methods 54 moderate skeletal fluorosis patients were randomly recruited into a treatment group and a control group. The treatment group was treated with Gun's MaQian decoction initially, and changed to be treated with GuKangNing capsule after 8 weeks' treatment. The control group was treated with placebo at first, and also changed to be treated with GuKangNing capsule after 8 weeks. Results Joint flexion and extension of elbow was improved greatly in both groups after the treatment, while the treatment group showing better effects than the control group (P<0.05). The improvement of joint flexion and extension of knee had not showed a statistical significance after the treatment. A 24 weeks follow-up to the joint flexion and extension of both elbow and knee revealed obvious improvement and the difference between the two groups were significance (P<0.05). Conclusion Gun's MaQian decoction can improve the flexion and extension of elbow and knee in moderate skeletal fluorosis patients.

OBJECTIVE: To present the experience of nasal reconstruction with forehead flap. METHOD: nasal reconstruction with forehead flap were applied in eight nasal carcinoma cases after operation and 5 nasal trauma cases with defects. RESULT: These forehead flaps were alive in all patients, all incision healed in I stage, no post operative complications were found. The shapes of nose were satisfactory, there were no recurrence of tumor during 1 to 17 year follow up. CONCLUSION The method can be clinically applied for its simple procedure, reliable flap's blood supply, high survival rate and satisfied result.
Adolescent ; Adult ; Aged ; Female ; Forehead ; surgery ; Humans ; Male ; Middle Aged ; Nose ; injuries ; surgery ; Rhinoplasty ; methods ; Skin Transplantation ; Surgical Flaps ; Young Adult

The teaching for medical advanced students is a routine and important task in teaching hospital.To increase the teaching quality,we should emphasize professional theory learning,increase clinical skill training,strengthen medical records writing training and enhance emotional exchange.