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*

β-hydroxy-β-methylbutyric acid (HMB) is widely applied in sports nutrition, disease prevention and other fields. However, chemical synthesis methods, limited by toxic reagents and violent reactions, can hardly meet the demands of green production. The biosynthesis method mainly utilizes enzymatic catalysis or metabolic engineering techniques for synthesis, and has the advantages of high efficiency, low cost, and sustainability. Therefore, the production of HMB by the biosynthesis method has a good application prospect. In this research, a biosynthesis-based production strategy for HMB was developed. By using L-leucine as the substrate and constructing a dual-enzyme co-expression system, we established an efficient catalytic process. At first, the enzymatic properties of L-amino acid deaminase (PvL-AAD) from Proteus vulgaris and 4-hydroxyphenylpyruvate dioxygenase (Rn4-HPPD) from Rattus norvegicus were characterized. Rn4-HPPD had low relative activity and required an acidic environment for catalysis. Based on the surface charge modification strategy of the enzyme protein, site-directed mutagenesis and combinatorial mutagenesis were conducted on 10 sites of Rn4-HPPD. A double mutant Rn4-HPPD^H18R/N302R was thus obtained, with the enzyme activities being 2.00 times and 2.39 times that of the wild type at pH 5.5 and pH 6.5, respectively. Subsequently, the expression of the two enzymes in Escherichia coli was optimized. After the optimal expression ratio of the two enzymes was determined as 1:3 and under the conditions of OD₆₀₀ of 70, pH 6.0, 35 ℃, Fe²⁺ concentration of 1.5 mmol/L, and feeding of the substrate in batches in a 5 L fermenter, the maximum yield of HMB reached 8.60 g/L. This study not only enhances the optimal pH and activity of Rn4-HPPD but also provides new approaches for the efficient microbial synthesis of HMB.
Escherichia coli/metabolism* ; Valerates/metabolism* ; Recombinant Proteins/biosynthesis* ; Animals ; Metabolic Engineering/methods* ; Rats ; Catalysis

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 explore the associated factors for membranous nephropathy (MN) patients with IgM deposition, and to construct a prediction model.Methods:This study was a retrospective cohort study. Patients diagnosed with MN with IgM deposition by renal biopsy in the Fourth Hospital of Hebei Medical University from February 2017 to December 2023 were retrospectively included. Clinical and pathological data were collected. The study population was randomized into a training set and a validation set at a 7:3 ratio. The endpoint event was defined as the remission of MN, and the patients were divided into remission group and non-remission group to compare the clinical and pathological examination results. Least absolute shrinkage and selection operator regression analysis and Cox regression analysis were used to explore the associated factors of poor prognosis of MN patients with IgM deposition. Internal validation was conducted using the validation set data. The clinical efficacy of the predictive model was evaluated by calculating the area under the receiver operating characteristic (ROC) curve and generating calibration curves. The total nomogram score for each patient was calculated based on the training set data, and the predictive performance was assessed by plotting the ROC curve. Patients were then stratified into low-risk and high-risk groups according to the optimal cut-off value derived from the ROC analysis of the total nomogram score. Kaplan-Meier survival analysis was performed to compare the remission rate between the two groups. Model performance was evaluated using the validation set.Results:A total of 200 MN patients with IgM deposition were included, and 49.0% of them achieved clinical remission. In the training set, statistically significant differences were observed in 24-hour urine protein quantification ( Z=-2.638, P=0.008), renal arteriolar wall thickening ( χ2=6.891, P=0.009), the proportion of patients receiving immunosuppressive therapy ( χ2=21.381, P<0.001), and the proportion of patients treated with corticosteroids combined with cyclophosphamide ( χ2=10.107, P=0.001). Through least absolute shrinkage and selection operator regression and Cox regression, 2 factors associated with clinical remission in MN patients with IgM deposition were simultaneously identified from 16 potential associated factors, including the use of immunosuppressants ( HR=3.823, 95% CI 2.055-7.113, P<0.001), and renal arteriolar wall thickening ( HR=0.428, 95% CI 0.221-0.831, P=0.012). Incorporating the clinical measurement of phospholipase A2 receptor (PLA2R) antibodies, a predictive model was established. The performance of the model was evaluated using the training dataset, yielding an area under the ROC curve of 0.731 (95% CI 0.648-0.814), with a sensitivity of 88.7% and a specificity of 55.1%. The optimal cut-off value was a total nomogram score of 41.7 points. The Kaplan-Meier survival analysis showed that the remission rate was significantly higher in the low-risk group than that of the high-risk group (Log-rank test, χ2=33.525, P<0.001). Model validation was performed using the validation dataset, which showed an AUC of 0.715 (95% CI 0.591-0.839), sensitivity of 70.4%, and specificity of 63.6%. Similarly, the Kaplan-Meier survival analysis demonstrated a significantly higher remission rate in the low-risk group than in the high-risk group (Log-rank test, χ2=8.467, P=0.004). Conclusion:A nomogram predictive model for remission of MN patients with IgM deposition, based on serum PLA2R antibody levels, the use of immunosuppressive therapy, and renal arteriolar wall thickening is developed. The model demonstrates a moderate clinical applicability.

Objective To review the state of research on medullary thyroid carcinoma(MTC)over the past two decades,so as to offer a quick reference for understanding the current state and future trends in this field.Methods A computer was employed to search the Web of Science Core Collection database for MTC-re-lated literature(types;articles and reviews)published between 2000 and 2022.VOS viewer 1.6.18 software was utilized to create collaboration network diagrams,heat distribution diagrams for high-yielding countries(with publication volume ≥ 90),research institutions(with publication volume ≥ 60),and authors(with publication volume ≥30),and high-frequency keyword clustering network diagrams.Cite Space 5.7.R5 soft-ware was employed to conduct cluster analysis and explosive keyword detection on co-cited literature.Results A total of 4866 MTC related literature(3688 articles and 918 reviews)were obtained.Although the annual publication volume fluctuated,the overall trend showed a gradual increase.The 4866 articles are from 87 coun-tries,involving 4250 research institutions,21 016 authors,and 963 journals.The United States(1436 arti-cles),MD Anderson Cancer Center at the University of Texas(139 articles),and Dralle H(Department of General Surgery at Halle Wittenberg University Hospital,61 articles)were the countries,research institu-tions,and authors with the highest number of publications,respectively.High frequency keywords form four clusters:RET gene expression,prognostic factors,MTC clinical management,and MTC treatment.The key-words emerging explosively in recent years were management,survival,guideline,association,etc.The key areas of focus in highly cited literature were RET proto oncogene,targeted therapy,RET gene mutation,and so on.Conclusions In the past 20 years,MTC related research has shown a gradual upward trend,with de-veloped countries led by the United States taking a leading position in this field.The current research hotspots are the treatment,diagnosis,application of inhibitors,and exploration of the pathogenesis of MTC.

Objective To investigate the association of alpha-fetoprotein(AFP)and prealbumin(PAB)with the 90-day prognosis of patients with hepatitis B virus-related acute-on-chronic liver failure(HBV-ACLF),as well as the difference in 90-day prognosis between the patients with different levels of AFP and PAB.Methods A total of 371 HBV-ACLF patients who were hospitalized in The General Hospital of Western Theater Command from January 2018 to January 2023 were enrolled,and according to the follow-up results on day 90 after discharge,they were divided into survival group with 216 patients and death group with 155 patients.The medical record system was used to collect general data,AFP,PAB,and other related laboratory markers.The t-test was used for comparison of normally distributed continuous data between two groups;a one-way analysis of variance was used for comparison between multiple groups,and the least significant difference t-test was used for comparison between two groups.The Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups,and the Kruskal-Wallis H test was used for comparison between multiple groups and further comparison between two groups.The chi-square test was used for comparison of categorical data between groups.The multivariate logistic regression analysis was used to identify the influencing factors for the prognosis of HBV-ACLF patients.The receiver operating characteristic(ROC)curve was plotted for AFP and PAB to determine their cut-off values.The Kaplan-Meier method was used to plot survival curves,and the Log-rank test was used for comparison.Results Compared with the death group,the survival group had significantly higher levels of hemoglobin(Hb),PAB,AFP,and platelet count(PLT)(all P<0.05)and significantly lower age,total bilirubin(TBil),white blood cell count(WBC),cystatin,creatinine,urea,international normalized ratio(INR),Model for End-Stage Liver Disease(MELD)score,proportion of patients with Child-Pugh class C,and incidence rates of ascites and hepatic encephalopathy(all P<0.05).The multivariate logistic regression analysis showed that PAB(odds ratio[OR]=0.985,95%confidence interval[CI]:0.972-0.998,P=0.024),AFP(OR=0.998,95%CI:0.996-1.000,P=0.028),PLT(OR=0.989,95%CI:0.982-0.996,P=0.003),age(OR=1.046,95%CI:1.018-1.075,P=0.001),TBil(OR=1.004,95%CI:1.002-1.006,P<0.001),and WBC(OR=1.237,95%CI:1.110-1.379,P<0.001)were independent influencing factors for 90-day prognosis in HBV-ACLF patients.According to the cut-off values of AFP and PAB on ROC curves,the patients were divided into group A with 102 patients(AFP≥73.19 ng/mL and PAB≥22.55 mg/L),group B with 170 patients(AFP≥73.19 ng/mL and PAB<22.55 mg/L;AFP<73.19 ng/mL and PAB≥22.55 mg/L),and group C with 99 patients(AFP<73.19 ng/mL and PAB<22.55 mg/L).There were significant differences between these three groups in age,Hb,INR,MELD score,and Child-Pugh class(all P<0.05).The Kaplan-Meier survival analysis showed that group A had a significantly higher 90-day cumulative survival rate than groups B and C(χ2=19.825,P<0.001).Conclusion AFP combined with PAB can better predict the 90-day prognosis of HBV-ACLF patients,and patients with high levels of AFP and PAB tend to have a lower mortality rate on day 90.

ObjectiveTo investigate the association of alpha-fetoprotein （AFP） and prealbumin （PAB） with the 90-day prognosis of patients with hepatitis B virus-related acute-on-chronic liver failure （HBV-ACLF）， as well as the difference in 90-day prognosis between the patients with different levels of AFP and PAB. MethodsA total of 371 HBV-ACLF patients who were hospitalized in The General Hospital of Western Theater Command from January 2018 to January 2023 were enrolled， and according to the follow-up results on day 90 after discharge， they were divided into survival group with 216 patients and death group with 155 patients. The medical record system was used to collect general data， AFP， PAB， and other related laboratory markers. The t-test was used for comparison of normally distributed continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for comparison between two groups. The Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups， and the Kruskal-Wallis H test was used for comparison between multiple groups and further comparison between two groups. The chi-square test was used for comparison of categorical data between groups. The multivariate logistic regression analysis was used to identify the influencing factors for the prognosis of HBV-ACLF patients. The receiver operating characteristic （ROC） curve was plotted for AFP and PAB to determine their cut-off values. The Kaplan-Meier method was used to plot survival curves， and the Log-rank test was used for comparison. ResultsCompared with the death group， the survival group had significantly higher levels of hemoglobin （Hb）， PAB， AFP， and platelet count （PLT） （all P<0.05） and significantly lower age， total bilirubin （TBil）， white blood cell count （WBC）， cystatin， creatinine， urea， international normalized ratio （INR）， Model for End-Stage Liver Disease （MELD） score， proportion of patients with Child-Pugh class C， and incidence rates of ascites and hepatic encephalopathy （all P<0.05）. The multivariate logistic regression analysis showed that PAB （odds ratio ［OR］=0.985， 95% confidence interval ［CI］： 0.972‍ ‍—‍ ‍0.998， P=0.024）， AFP （OR=0.998， 95%CI： 0.996‍ ‍—‍ ‍1.000， P=0.028）， PLT （OR=0.989， 95%CI： 0.982‍ ‍—‍ ‍0.996， P=0.003）， age （OR=1.046， 95%CI： 1.018‍ ‍—‍ ‍1.075， P=0.001）， TBil （OR=1.004， 95%CI： 1.002‍ ‍—‍ ‍1.006， P<0.001）， and WBC （OR=1.237， 95%CI： 1.110‍ ‍—‍ ‍1.379， P<0.001） were independent influencing factors for 90-day prognosis in HBV-ACLF patients. According to the cut-off values of AFP and PAB on ROC curves， the patients were divided into group A with 102 patients （AFP≥73.19 ng/mL and PAB≥22.55 mg/L）， group B with 170 patients （AFP≥73.19 ng/mL and PAB<22.55 mg/L； AFP<73.19 ng/mL and PAB≥22.55 mg/L）， and group C with 99 patients （AFP<73.19 ng/mL and PAB<22.55 mg/L）. There were significant differences between these three groups in age， Hb， INR， MELD score， and Child-Pugh class （all P<0.05）. The Kaplan-Meier survival analysis showed that group A had a significantly higher 90-day cumulative survival rate than groups B and C （χ²=19.825， P<0.001）. ConclusionAFP combined with PAB can better predict the 90-day prognosis of HBV-ACLF patients， and patients with high levels of AFP and PAB tend to have a lower mortality rate on day 90.

Thyroid cancer is the only non reproductive tumor that mainly occurs in women.The morbidity rate of female thyroid cancer is three times higher than that of male thyroid cancer.However,the ex-isting studies have not systematically summarized the factors that contribute to the high incidence of thyroid cancer in women.This paper reviews all the literatures that mention the high incidence of thyroid cancer in women,and sorts out the main reasons,such as estrogen,obesity,reproductive factors,oxidation antioxidant system imbalance,X chromosome,etc.,which are the main reasons for the current research finding that the in-cidence rate of thyroid cancer in women is higher than that in men.This paper makes a vivid graphic analysis and summary of the reasons,providing a research direction for future researchers to research on gender duality of thyroid cancer,and providing a certain reference for the clinical diagnosis and treatment of gender differences in thyroid cancer.

Objective To explore the effect of community pharmacy outpatient service on patients with type 2 diabetes mellitus. Methods A non-randomized controlled study was conducted, and type 2 diabetes patients managed in the community were divided into an intervention group of 112 cases and a control group of 110 cases. The control group received routine medication guidance during general practice outpatient visits, while the intervention group received comprehensive pharmacy outpatient service intervention based on routine medication guidance in general practice. Follow-up visits were conducted every 3 months. Repeated measurement analysis of variance and multivariate linear regression analysis were used to evaluate the intervention effect of the pharmacy outpatient service. Results Fasting blood glucose and glycosylated hemoglobin levels in the intervention group showed a decreasing trend with the increase of intervention time compared to pre-intervention time （P<0.01）, with increased duration of weekly exercise, decreased staple food intake, increased vegetable intake, and increased medication adherence score （P<0.01）. After adjusting for confounding factors through multivariate linear regression model, pharmacy outpatient intervention was found to be an independent protective factor for fasting blood glucose level （β=−0.891, P<0.01） and glycosylated hemoglobin level （β=−0.760, P<0.01） in the study subjects. Conclusion The community pharmacy outpatient service could enhance the self-management ability of patients with type 2 diabetes mellitus, and effectively improve patients’ fasting blood glucose and glycosylated hemoglobin.

Objective To examine the association between lymphocyte subsets and renal clinicopathological characteristics as well as prognosis in patients with IgA nephropathy(IgAN).Methods The retrospective analysis included general clinical data and pathological examination results of IgAN patients diagnosed by renal biopsy at the Fourth Hospital of Hebei Medical University from January 2018 to January 2022.Correlation tests were conducted to examine the relationship between lymphocyte subsets and other significant clinicopathological parameters.The optimal cut-off value of CD4+T determined using the Youden index,and patients were grouped accordingly.Kaplan-Meier survival curves and Cox regression analyses were employed to compare the low and high CD4+T lymphocyte groups among IgAN patients,identifying factors influencing renal function progression.The endpoint event was defined as a decrease in estimated glomerular filtration rate(eGFR)of≥30%from baseline,progression to end-stage renal disease(ESRD)[eGFR<15 mL/(min·1.73 m2)or initiation of renal replacement therapy],or all-cause mortality.Results Low CD4+T lymphocytes were significantly positively correlated with blood IgA levels and the proportion of glomerular crescents in IgAN patients(all P<0.05).This study included a total of 53 IgAN patients,divided into two groups based on CD4+T lymphocyte counts:20 patients in the low CD4+T lymphocyte group and 33 patients in the high CD4+T lymphocyte group.In the low CD4+T lymphocyte group,there was a higher proportion of males and a lower proportion of glomerular crescents(P<0.05).Kaplan-Meier survival analysis revealed that patients with low CD4+lymphocytes had a significantly lower cumulative renal survival rate(Log-Rank test χ2=4.188,P=0.041).Cox regression analysis indicated that low CD4+lymphocytes were an independent risk factor for the progression of renal function decline in IgAN patients(HR=2.614,95%CI:1.006～6.788,P=0.048).Conclusions Patients with higher levels of CD4+T lymphocytes exhibit a lower risk of adverse renal outcomes.In contrast,patients with IgA nephropathy and low CD4+T lymphocyte counts tend to have poorer renal survival rates.