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 assess the efficacy of neoadjuvant treatment with PD-1 (programmed cell death protein 1) inhibitors combined with paclitaxel (albumin-conjugated) and cisplatin (TP regimen) for locally advanced hypopharyngeal squamous cell carcinoma and laryngeal organ function preservation.Methods:Data of 53 patients, including 51 males and 2 females, aged 38-70 years old, who were diagnosed with locally advanced hypopharyngeal squamous carcinoma confirmed by histology and enhanced CT at the Cancer Prevention and Control Center of Sun Yat-sen University during the initial treatment from January 1, 2019 to January 15, 2023, were retrospectively analyzed. All patients received neoadjuvant therapy with PD-1 inhibitors combined with albumin-bound paclitaxel (260 mg/m 2) and cisplatin (60 mg/m 2) for 3 to 4 cycles. The main outcome measures were larynx dysfunction-free survival (LDFS), overall survival (OS), and progression-free survival (PFS). Survival curves were plotted using the Kaplan-Meier method, and Cox multifactorial analysis was further performed if Cox univariate analysis was statistically significant. Results:The overall efficiency was 90.6% (48/53). The 1-year and 2-year LDFS rates were 83.8% (95% CI: 74.0% to 94.8%) and 50.3% (95% CI: 22.1% to 91.6%), the 1-year and 2-year OS rates were 95.2% (95% CI: 88.9% to 100.0%) and 58.2% (95% CI: 25.6% to 81.8%), and the 1-year and 2-year PFS rates were 83.9% (95% CI: 74.2% to 94.9%) and 53.5% (95% CI: 32.1% to 89.1%). Adverse events associated with the neoadjuvant therapy were mainly myelosuppression (45.3%), gastrointestinal reactions (37.7%) and hypothyroidism (20.8%). Conclusion:The neoadjuvant treatment of locally advanced hypopharyngeal squamous cell carcinoma using PD-1 inhibitors combined with paclitaxel and cisplatin can provide with a higher survival rate with a improved laryngeal organ function preservation rate.

Objective The imaging findings of X-ray defecography(XRD)and magnetic resonance defecog-raphy(MRD)of patients with Solitary Rectal Ulcer syndrome(SRUS)were retrospectively analyzed to provide important information for clinical diagnosis and treatment.Methods 19 patients with SRUS confirmed by clinical,pathological and colonoscopy were included in this study.Among them,15 patients underwent XRD and 7 patients underwent MRD,and 3 patients underwent both XRD and MRD.Data of all enrolled patients were collected and pelvic floor function was measured.Results In the results of XRD,3 patients(20%)showed rectal intussusception.8 patients(53.3%)showed external rectal prolapse and 2 patients(13.3%)showed moderate rectocele.In addition,there were 2 patients of puborectal muscle hypertrophy,and 1 patient of bladder prolapse and uterine prolapse,respectively.For MRD,3 patients(42.9%)showed rectal mucosal prolapse(partial prolapse).At 4 patients(57.1%)with rectocele,3 patients(all female)had moderate rectocele,1 patient had mild rectocele.3 patients were also observed related anterior and middle compartment organ descent.2 patients of pubulorectal muscle hypertrophy,no sigmoidocoele.Conclusion Defecography can evaluate the structural and functional abnormalities of pelvic floor in SRUS patients,such as external rectal prolapse,rectal protrusion,rectal mucosal prolapse,and rectal intussusception,which has guiding significance for the treatment of SRUS patients.

Objective To investigate the value of combining preoperative magnetic resonance imaging(MRI)and postoperative pathological assessment of lymph node metastasis in predicting overall survival in rectal cancer patients.Methods This retrospective study collected clinical,pathological and image information of 2610 patients histopathologically confirmed with rectal adenocarcinoma at the Sixth Affiliated Hospital of Sun Yat-sen University between January 2016 and December 2021.All patients underwent MRI scans and were divided into three groups according to lymph node status assessed by preoperative MRI(MRIN)and postoperative pathology(PN):MRIN+but PN-(MRIN+group),PN+but MRIN-(PN+group),MRIN+and PN+(MRI-PN+group).Inverse probability weighting(IPW)was used to adjust for confounding factors.Kaplan-Meier curves were used to estimate overall survival and log-rank tests were used to compare the difference.Univariate Cox regression models were used to analyze the correlation between tumor characteristics and overall survival,and bidirectional stepwise Cox regression models were used to identify independent risk factors for overall survival.Results The MRI-PN+group showed higher tumor staging,more frequent perineural invasion,more distant metastases,and a higher risk of death compared to the P N+group and MRIN+group(all P＜0.05).Kaplan-Meier curves showed that the 3-year survival rates for the MRIN+group,PN+group,and MRI-PN+group were 90.5%,79.1%,and 76.4%,respectively;the 5-year survival rates were 85.7%,71.5%,and 59.2%,respectively.Stepwise Cox regression showed that age,tumor location,carcino-embryonic antigen,carbohydrate antigen 19-9,lymph nodes number,pathological tumor stage,lymphovascular invasion,perineural invasion,distant metastasis,neoadjuvant therapy and adjuvant therapy,and MRI-pathology lymph node status were independent risk factors for overall survival in rectal cancer(all P＜0.05).Conclusion Evaluating the lymph node status by combining preoperative MRI and postoperative pathology helps predict overall survival in rectal cancer patients more accurately.

Objective:To observe the epidemiology, clinical manifestations, laboratory tests, imaging findings, treatment and prognosis of patients with corona virus disease 2019.Methods:Clinical data of 109 patients with suspected and definite corona virus disease 2019 admitted to the Sixth Hospital of Wuhan from December 24, 2019 to January 28, 2020 were retrospectively analyzed. Statistical analysis was performed by using t test or chi-square test. Results:Among the 109 patients, 54(49.5%) patients had definite contact history. Among the 109 patients, 104(95.4%) presented with fever, 37(33.9%) with headache, 78(71.6%) with general pain, 88(80.7%) with fatigue and poor appetite, 23(21.1%) with diarrhea, 94(86.2%) with coughing, 23(21.1%) with shortness of breath, 57(52.3%) with palpitation, 45(41.3%) with chest distress, 4(3.7%) with chest pain, 40(36.7%) with lung rales. Forty-two cases (38.5%) had leukocyte count <4×10 9/L, 58 cases (53.2%) had lymphocyte count <1.5×10 9/L, 27 cases (24.8%) had hemoglobin <120 g/L, 37 cases (33.9%) had lactic dehydrogenase (LDH) >230 mmol/L, 29 cases (26.6%) had pro-brain natriuretic peptide>300 ng/mL, 87 cases (79.8%) had hypersensitive C reactive protein>10 mg/L, 26 cases (23.9%) had D-dimer>0.5 mg/L, 35 cases (32.1%) had coagulation disorder. On admission, chest computed tomography showed that 27 cases (24.8%) of pneumonia were unilateral, 82 cases (75.2%) were bilateral, and most of them were ground glass. The leukocyte counts, LDH, pro-brain natriuretic peptide and D-dimer of severe/critical cases ((11.33±4.87)×10 9/L, (527.51±260.87) mmol/L, (722.88±189.56) μg/L, (4.24±1.89) mg/L, respectively) were all higher than those of common cases ((4.02±1.49)×10 9/L, (159.75±30.31) mmol/L, (428.22±124.76) μg/L and (0.41±0.22) mg/L, respectively), while the lymphocyte count of severe/critical cases ((0.60±0.17)×10 9/L) was lower than common cases ((1.13±0.43)×10 9/L) ( t=11.36, 11.33, 9.81, 2.81 and 7.77, respectively, all P<0.05). The comprehensive treatment included antiviral drugs, prevention of bacterial infection and supportive treatment, and glucocorticoid and respiratory support treatment were administrated when necessary. Conclusions:The corona virus disease 2019 is characterized by highly infectious, rapid progression, and diverse clinical and imaging features. Early diagnosis and active comprehensive treatment could improve the prognosis and reduce the mortality.

OBJECTIVE:To provide reference for promoting the management of antibiotics in the hospitals. METHODS:Each 3 hospitals from county second grade class A general hospitals,municipal third grade class A general hospitals and provincial third grade hospitals(including 2 third grade class A general hospitals and one third grade special hospital)in Anhui province were select-ed and respectively included into group A,B,and C. By on-site survey mode,questionnaires were used to investigate and score the situation of antibiotics management in hospitals of 3 groups,and then analyzed statistically. RESULTS:Scoring rate (SR) of overall situation on antibiotics management were 65.33%,89.34% and 86.67% in group A,B and C respectively;group A was statistically significantly lower than group B and C (P<0.001). SR of basic situation indicator on antibiotics management were 90.46%,95.25% and 95.25% in group A,B and C,respectively and there was no statistically significant difference among 3 groups (P>0.05). SR of development indicator of antibiotics management were 58.33%,88.90% and 86.10%,respectively in group A,B and C;group A was significantly lower than group B and C (P<0.001). SR of information construction indicator of antibiotics management were 50.00%,83.33% and 77.79% respectively in group A,B and C;group A was significantly lower than group B and C (P≤0.001). SR of information construction indicator of antibiotics management in 3 groups were all signifi-cantly lower than SR of basic situation indicator(P<0.05). SR of development indicator of antibiotics management in group A and C were all significantly lower than SR of basic situation indicator(P<0.05). CONCLUSIONS:The antibiotics management of mu-nicipal third grade class A general hospital is equal to that of provincial third grade hospital,and that of county second grade class A general hospital is in relatively low level. Different levels of hospitals should further strengthen the related antibiotics manage-ment work and information construction,especially for county second grade class A general hospitals.

Objective To investigate the effect of Azithromycin combined with terbutaline on the treatment of pneumonia in children and its effect on symptom improvement.MethodsThe clinical data of children with pneumonia treated in Taizhou Hospital of traditional Chinese and Western medicine from January 2014 to December 2016 were retrospectively analyzed, according to the treatment methods,the patients were divided into control group and observation group, the control group was given azithromycin treatment, the observation group on this basis to give terbutaline atomization treatment.The therapeutic effects of the two groups were observed, and the differences of clinical symptom score, inflammatory factor level were compared between the two groups before and after treatment.ResultsThe effective rate of the observation group was 98%, which was significantly higher than that of the control group (84%);The clinical symptom score had no difference between the two groups before treatment, after treatment, the observation group of cough, expectoration, fever and rales were lower than those in the control group;The two groups had no difference between the levels of proinflammatory cytokines, after treatment, the observation group IL-6, IL-8, TNF-and CRP levels were lower than the control group.ConclusionAzithromycin combined with terbutaline in the treatment of children with pneumonia has a better therapeutic effect, can significantly improve the clinical symptoms, reduce the level of inflammatory factors, has a good clinical value.

Objective To evaluate the value of N-terminal prohormone brain natriuretic peptide (NT-proBNP) and tissue Doppler imaging (TDI) in patients with left ventricular diastolic dysfunction.Methods Forty-three patients with diastolic heart failure were enrolled. Pulsed wave (PW) Doppler technology was used to detect early diastolic mitral velocity (E) and TDI was performed to determine the mitral annulus velocity of early diastolic phase (Em). According to the E/Em, patients were divided into three groups: group Ⅰ (E/Em ＜8),group Ⅱ (E/Em 8-15) and group Ⅲ (E/Em ＞ 15). The plasma NT-proBNP was measured. Results The plasma NT-proBNP in group Ⅲ [( 1119 ± 3123) ng/L] was higher than that in group Ⅱ [(684± 1742) ng/L] and group Ⅰ [(276±438) ng/L] (P＜0.01),and the plasma NT-proBNP in group Ⅱ was higher than that in group Ⅰ (P ＜ 0.01 ). The plasma NT-proBNP had positive correlation with E/Em (r = 0.438,P ＜ 0.01 ). Conclusion The plasma NT-proBNP and TDI are useful to evaluate the left ventricular diastolic dysfunction.