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 compare the efficacy of hepatic artery infusion chemotherapy (HAIC) combined with targeted therapy and immunnotherapy and transarterial chemoembolization (TACE) combined with targeted therapy and immunnotherapy in the treatment of unresectable hepatocellular carcinoma HCC.Methods:We retrospectively analyzed the clinical data of 40 patients with unresectable HCC treated with HAIC combined with targeted therapy and immunnotherapy and TACE combined with targeted therapy and immunnotherapy in Tongde Hospital of Zhejiang Province and Zhejiang Provincial People's Hospital.The patients were divided into HAIC group ( n=14) and TACE group ( n=26) according to the different treatment methods. Baseline data, surgical conversion and intraoperative situation, tumor response, portal vein cancer thrombus control rate, leukocyte reduction rate, platelet reduction rate, incidence of liver function abnormalities, objective remission rate, and disease control rate were compared between the two groups. Results:The HAIC group had a later baseline tumor staging than the TACE group (higher percentage of portal vein cancer thrombus, CNLC stage Ⅲa).The surgical conversion rate of the HAIC and TACE groups were 28.6%(4/14) and 26.9%(7/26), respectively, with the difference of no statistical significance ( P>0.05);The operation time and intraoperative bleeding were (329.5±19.9) min vs.(413.4±26.4) min, (272.2±49.9) ml vs.(536.0±123.6) ml, and the differences were statistically significant ( P<0. 05); The maximum tumor diameter reduction rate [(30.7%±15.1%) vs.(7.2%±12.6%)] and portal vein cancer thrombus control rate [100% (12/12) vs. 64.3% (9/14)], the differences were statistically significant ( P<0.05);The incidences of leukocyte and platelet decrease in the two groups during the course of treatment were 71.4%(10/14) vs. 34.6%(9/26)、78.5%(11/14) vs. 38.5%(10/26), and the incidences of liver function abnormalities were 35.7%(5/14) vs. 69.2%(18/26), the differences were statistically significant ( P<0.05);The objective response rate and disease control rate were 57.1%(8/14) vs. 30.8% (8/26)、71.4% (10/14) vs. 53.8%(14/26), all statistically significant. Conclusion:HAIC combined with targeted therapy and immunnotherapy is a safe and effective treatment for middle and advanced HCC, especially suitable for patients with portal vein tumor thrombus(PVTT), large tumor, or poor liver function.

Objective To investigate the effects of Bushen Zhuanggu decoction on total N-terminal propeptide of type Ⅰprocollagen(t-PINP)and β-C-terminal telopeptide of type Ⅰ collagen(β-CTX)in patients with knee osteoarthritis(KOA).Methods A total of 120 patients with liver-kidney deficiency KOA treated in Linping District Hospital of Traditional Chinese Medicine from June 2021 to April 2024 were selected and divided into study group and control group according to random number table method,with 60 cases in each group.The patients of control group was given oral treatment with etocoxib and omeprazole,and patients of study group was given oral treatment of Bushen Zhuanggu decoction on the basis of control group.Both groups were treated for one month.The clinical efficacy,TCM syndrome scores,serum t-PINP and β-CTX leves,t-PINP/β-CTX,Western Ontario and Mcmaster Universities osteoarthritis index(WOMAC)scores and adverse reactions during treatment were compared between two groups.Results The total effective rate in study group was significantly higher than that in control group(x2=4.5 13,P=0.034).After treatment,the TCM syndrome scores,serum t-PINP and β-CTX levels and WOMAC scores in two groups were significantly lower than before treatment,and t-PINP/β-CTX were significantly higher than before treatment(P＜0.05).The TCM syndrome scores,serum t-PINP and β-CTX levels and WOMAC scores in study group were significantly lower than those in control group,and t-PINP/β-CTX was significantly higher than that in control group(P＜0.05).There was no significant difference in incidence of adverse reactions between two groups(x2=0.686,P=0.408).Conclusion Bushen Zhuanggu decoction can improve the clinical efficacy of KOA patients with liver-kidney deficiency,relieve clinical symptoms and pain,increase t-PINP/β-CTX,and reduce the incidence of adverse reactions.

Objective:To analyze the drug-resistance pattern and molecular epidemiological characteristics of Enterobacteriales carrying the blaNDM gene in Lishui, aiming to guide clinical anti-infection treatment. Methods:Non-duplicate blaNDM-carrying Enterobacteriales, isolated from Lishui Central Hospital, were collected and identified by VITEK MS. The minimal inhibitory concentrations (MICs) were detected by the broth microdilution method. The ST types of the strains were determined by multilocus sequence typing (MLST). Plasmid types were identified by transformation or conjugation experiments and replication initiator amplification experiments. The transposon structures were detected by PCR amplification. Finally, the epidemic regularity of blaNDM gene in Lishui was analyzed from three levels: clonal group, plasmid, and mobile genetic elements. Results:A total of 109 blaNDM-positive strains were collected. Among them, 60 strains carried the blaNDM-1 gene and 49 strains carried the blaNDM-5 gene. The 109 strains showed 100% resistance to ceftazidime and cefotaxime. The resistance rates to peracillin-tazobactam and imipenem were higher than 80%. Strains carrying the blaNDM-5 gene were more resistant to meropenem than those carrying blaNDM-1 gene( P<0.05). A total of 68 STs were detected from 109 strains, and IncX3, IncFⅡγ, IncA/C and IncT/R plasmids were detected, and 90.83% of the blaNDM genes were located in the IncX3 plasmid. Twelve types of blaNDM gene surrounding structures existed, and they all carried the highly conserved blaNDM- bleMBL- trpF gene sequence. Conclusions:The blaNDM gene has diverse transmission modes in Lishui. The IncX3 plasmid is the main factor mediating its transfer, and all strains carry highly conserved blaNDM- bleMBL- trpF gene sequence.

The importance of gait assessment in the rehabilitation of cancer patients is gradually being recognized. However, quantitative analysis of balance ability in cancer patients is still limited. A total of 102 cancer patients meeting the inclusion criteria were recruited from Hefei Cancer Hospital, Chinese Academy of Sciences. Their balance ability was evaluated using the Berg Balance Scale (BBS). Gait data were collected by an electronic walkway and an IMU sensor system, including spatial-temporal and kinematic gait features such as step length, cadence, support time, and range of motion. Recursive feature elimination was used for feature selection. Ridge, Elastic Net, SVR, RF, and AdaBoost models were used to predict balance ability scores. Five-fold cross-validation was used to evaluate the performance of these models. Results show that the SVR model achieves the best performance with fifteen features (RMSE=3.22, R ²=0.91), followed by Ridge (RMSE=3.63, R ²=0.89). A method for evaluating balance ability based on gait features is proposed, providing a quantitative tool for personalized rehabilitation interventions in cancer patients.
Humans ; Postural Balance ; Neoplasms/rehabilitation* ; Gait ; Gait Analysis ; Biomechanical Phenomena ; Female

BACKGROUND: The mechanisms distinguishing metabolically healthy from unhealthy phenotypes within the same BMI categories remain unclear. This study aimed to investigate the associations between regional fat distribution and metabolically unhealthy phenotypes in Chinese adults across different BMI categories. METHODS: This cross-sectional study involving 11833 Chinese adults aged 20 years and older. Covariance analysis, adjusted for age, compared the percentage of regional fat (trunk, leg, or arm fat divided by whole-body fat) between metabolically healthy and unhealthy participants. Trends in regional fat percentage with the number of metabolic abnormalities were assessed by the Jonckheere-Terpstra test. Odds ratios (ORs) and their 95% confidence intervals (CIs) were estimated by logistic regression models. All analyses were performed separately by sex. RESULTS: In non-obese individuals, metabolically unhealthy participants exhibited higher percent trunk fat and lower percent leg fat compared to healthy participants. Additionally, percent trunk fat increased and percent leg fat decreased with the number of metabolic abnormalities. After adjustment for demographic and lifestyle factors, as well as BMI, higher percent trunk fat was associated with increased odds of being metabolically unhealthy [highest vs. lowest quartile: ORs (95%CI) of 1.64 (1.35, 2.00) for men and 2.00 (1.63, 2.46) for women]. Conversely, compared with the lowest quartile, the ORs (95%CI) of metabolically unhealthy phenotype in the highest quartile for percent arm and leg fat were 0.64 (0.53, 0.78) and 0.60 (0.49, 0.74) for men, and 0.72 (0.56, 0.93) and 0.46 (0.36, 0.59) for women, respectively. Significant interactions between BMI and percentage of trunk and leg fat were observed in both sexes, with stronger associations found in individuals with normal weight and overweight. CONCLUSIONS Trunk fat is associated with a higher risk of metabolically unhealthy phenotype, while leg and arm fat are protective factors. Regional fat distribution assessments are crucial for identifying metabolically unhealthy phenotypes, particularly in non-obese individuals.
Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Young Adult ; Adipose Tissue ; Body Fat Distribution ; Body Mass Index ; China/epidemiology* ; Cross-Sectional Studies ; Health Surveys ; Phenotype

As an intermediate phenotype for multiple cardiovascular diseases, left ventricular hypertrophy (LVH) benefits from early diagnosis, which allows for timely intervention to prevent worsening of the condition, mitigate severe complications like heart failure and arrhythmias, and consequently improve patient outcomes. Preliminary advances have been made using deep learning for the early diagnosis and identification of etiology in LVH. This paper reviews the pathophysiology, causes, and diagnostic standards for LVH, discusses the strengths and weaknesses of applying deep learning to diagnostic tools such as echocardiography, cardiac magnetic resonance imaging, and electrocardiogram, examines its use in prognostic evaluation, and concludes by summarizing current achievements and suggesting future research avenues.

Objective To investigate the impact of various health education intervention strategies on the proper use of personal respiratory protective equipment (RPE) among workers exposed to dust. Methods Dust-exposed workers were recruited from 60 selected enterprises in Guangdong Province using cluster random sampling method. They were randomly allocated to the control, low-intensity intervention, and high-intensity intervention groups, with 358, 346, and 371 workers in each group, respectively. Workers in the control group received no designed intervention. Workers in the low-intensity intervention group received traditional plus mobile health education on the proper use of RPE. Workers in the high-intensity intervention group received all components of the low-intensity intervention, supplemented with peer education. The intervention lasted for six months. RPE usage was compared among the three groups of workers before and after the intervention. Results Workers in the control, low-intensity intervention, and high-intensity intervention groups showed higher rates of both RPE wearing and correct RPE wearing after the intervention than before it within their respective groups (RPE wearing rate: 94.1% vs 99.2%, 95.7% vs 100.0%, 94.6% vs 100.0%, all P<0.01; correct RPE wearing rate: 66.8% vs 91.1%, 67.3% vs 95.7%, 66.6% vs 96.5%, all P<0.01). Post-intervention correct RPE wearing rates were highest in the high-intensity intervention group, followed by the low-intensity intervention group, and the control group, with the percentage of 96.50%, 95.66% and 91.06%, respectively (P<0.01). Binary logistic regression analysis result showed that different intervention strategies affected the correct use of personal RPE among dust-exposed workers after adjusting for gender, age, and other confounding factors (P<0.05). Compared with the control group, the rates of correct RPE use increased in the low-intensity intervention group and the high-intensity intervention group (odd ratio was 2.14 and 3.01; 95% confidence interval was 1.12 - 4.10 and 1.53 - 5.91, respectively). Conclusion The implementation of traditional plus mobile health education interventions on the proper use of RPE can promote correct RPE utilization among dust-exposed workers, and integrating peer education further enhances the intervention effectiveness.