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*

Objective : To survey the current situation and analyze the relevant influencing factors of sleep quality in adolescent patients with temporomandibular joint anterior disc displacement. Methods: This study has been reviewed and approved by the Ethics Committee, and informed consent has been obtained from patients. A convenience sampling method was used to investigate 120 adolescent patients with temporomandibular joint anterior disc displacement in the outpatient department of stomatology in a grade A tertiary hospital in Shanghai using the general data questionnaire, the Pittsburgh sleep quality index scale (PSQI), the Chinese middle school student mental health scale (MMHI-60) and the pain visual analog scale (VAS). Descriptive analysis, single factor analysis, correlation analysis and multiple regression analysis were used to explore the relevant influencing factors. Results: The PSQI score of adolescent patients with temporomandibular joint anterior disc displacement in this study was 7.77 ± 4.63. There was a statistically significant difference in sleep quality among patients with different academic pressures and levels of sleep bruxism (P<0.05). The sleep quality score was positively correlated with the pain score (r = 0.45, P<0.001) and positively correlated with the psychological score (r = 0.74, P<0.001). The degree of pain can affect the patient's sleep quality, and those with good mental health have better sleep quality. The results of regression analysis showed that academic stress (OR = 2.511, 95% CI =1.307 ~ 4.828), bruxism (OR = 3.694, 95% CI = 1.394 ~ 9.791), pain score (OR = 2.104, 95% CI =1.095 ~ 4.041) and psychological score (OR = 1.039, 95% CI = 1.021 ~ 1.058) were statistically significant. Conclusion The sleep quality of adolescent patients with temporomandibular joint anterior disc displacement is generally poor. Academic pressure, sleep bruxism, pain and mental health are the influencing factors of sleep quality.

Objective To assess alternations in left ventricular volume and systolic synchrony in patients with frequent premature ventricular complexes(PVCs) from the right ventricular outflow tract(RVOT).Methods Twenty-nine patients with frequent isolated PVCs from RVOT were included and 30 healthy subjects as control.Instantaneous full-volume imaging(IFI) was performed to evaluate left ventricle volumetric parameters,including end-systolic volume (ESV),end-diastolic volume (EDV),stroke volume (SV),ejection fraction (EF),and systolic synchrony parameters,including systolic dyssynchrony index (SDI),dispersion end-systole (DISPES),mean end-systolic time (MES),pre-contraction time volume (PreContr) and post-contraction time volume (PostContr).Contraction front mapping was performed to visualize volumetric contraction sequence.All values of patients with PVCs were recorded during sinus beats (PVC-S) and premature ventricular beats (PVC-V) respectively.Results Significant differences were observed in left ventricular systolic volumetric and synchrony parameters between PVC-V and control subjects (P＜0.01),as well as in MES and PreContr between PVC-S and control subjects (P＜0.01).Conclusions Left ventricular systolic dysynchrony was demonstrated in patients with PVCs from RVOT.IFI was a novel tool to analyze left ventricular global and regional volumetric alternations.

Objective To investigate the histogenesis,clinicopathological features and biological behavior of goblet cell carcinoid (GCC) of the appendix. Methods 3 cases of GCC of the appendix were studied by light microscopy and immunohistochemistry. Results 3 cases all occured in men. Microscopically: the tumor was composed of goblet cells containing mucin-filled with basal round to crescentic nuclei without atypia.These tumor cells were arranged in single forms,nests or acinus with no central lumen. 1 case was composed of simple goblet cells and typical carcinoid cells were seen in other 2 cases arranged in trabeculae and tubule.There was transition between goblet cells with tubelar and crypt epithelium.Immunohistochemical staining revealed that goblet cells were positively reacted with CgA, Syn, CEA, CK and p53 in 1 case. Conclusions Goblet cell carcinoid of the appendix arise from a pluripotent cell with divergent neuroendocrine and mucinous differentiation .It is a subtype of carcinoids of the appendix.The diagnosis mainly bases on its morphologic changes and immunohistochemical findings. The unpredictable behavior of this tumor is probably related to its component and the degree of infiltration.