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.

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.

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

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.

Objective:To investigate the effect of maternal serum vitamin D on fetal long bone development.Methods:A retrospective collection of 1 193 first-time pregnant women who visited our hospital′s prenatal diagnosis center from July 2018 to June 2020 was conducted. All underwent prenatal fetal ultrasound examination and serum vitamin D level detection. Based on the dosage of vitamin D administered after the first vitamin D test, participants were divided into the basic dosage group(vitamin D 3, 1 600 IU/d, orally) and the adequate supplementation group(vitamin D 2 injection 600 000 IU/2 weeks, intramuscular injection). The serum 25-(OH)D levels of the two groups of pregnant women were compared at 12 and 24 weeks of treatment, as well as the long bone growth of their fetuses. Multivariable logistic regression analysis was used to analyze the factors influencing fetal long length. Results:Compared to the basic dosage group, the adequate supplementation group showed a significant increase in serum 25-(OH)D levels in pregnant women at 8 weeks, 12 weeks, and 24 weeks of treatment. The adequate supplementation group also significantly increased fetal long bone length at 12 weeks[(4.93±0.75) cm vs(4.61±0.73) cm, P<0.05] and 24 weeks of treatment [(7.92±0.84) cm vs(7.25±0.92) cm, P<0.05], with the difference between the two groups being more pronounced at 24 weeks of treatment. Maternal height, basal vitamin D level, and vitamin D level at 24 weeks of gestation were positively correlated with fetal long bone length. Conclusion:Pregnant women should maintain a relatively high level of basal vitamin D, and pay attention to the effect of vitamin D level on the fetus. A sufficient amount of vitamin D supplementation is of great significance for the long bone development of the fetus.

ObjectiveTo explore the comprehensive effects of Qingxin Zishen decoction on the symptom score and neuroendocrine indexes and the mechanism of the decoction in regulating KNDy neurons in the patients with menopausal syndrome. MethodA total of 60 patients with menopausal syndrome due to yin deficiency with effulgent fire who attended the menopausal outpatient of Jiangsu Province Hospital of Chinese Medicine were randomized into an experimental （Qingxin Zishen decoction） group （30 cases） and a control （femoston） group （30 cases）. The treatment lasted for 12 weeks in both groups. The two groups were compared in terms of the comprehensive efficacy， frequency and degree of hot flashes and sweating， modified Kupperman score， and the serum levels of hypothalamic peptide kisspeptin， neurokinin B （NKB）， dynorphin （Dyn）， follicle-stimulating hormone （FSH）， and estradiol （E₂）. Result① Comprehensive efficacy： The comprehensive efficacy of the two groups was comparable. ② Frequency and degrees of hot flashes and sweating： After treatment， the frequency and degrees of hot flashes and sweating in the two groups were reduced （P<0.05） and the control group outperformed the experimental group （P<0.05）. ③ Modified Kupperman score and menopausal symptoms： After treatment， the modified Kupperman score decreased in both groups （P<0.05）. After 4 weeks of treatment， the experimental group was superior to the control group in terms of the scores of dizziness and headache （P<0.05）. ④ Serum levels of sex hormones： After treatment， the serum E₂ level elevated and the FSH level lowered in both groups （P<0.05）， and the changes were more obvious in the control group （P<0.05）. ⑤ Neuroendocrine indexes： After treatment， the serum levels of kisspeptin and NKB in the two groups decreased （P<0.05）， and the serum Dyn level in the experimental group increased （P<0.05）. Moreover， the experimental group had higher Dyn level than the control group after treatment （P<0.05）. ConclusionQingxin Zishen decoction can alleviate hot flashes， sweating， and other symptoms in the women with menopausal syndrome by acting on the KNDy neurons to lower the kisspeptin and NKB levels and elevate the Dyn level. The findings provide new ideas for the clinical treatment of hot flashes in menopause.

Objective To investigate the diagnostic efficacy and clinical value of GNB4 and Riplet gene methylation alone and in combination in the diagnosis of primary liver cancer.Methods A total of 313 patients were selected,including 78 patients with primary liver cancer,41 patients with other digestive system tumors,17 patients with non-digestive system tumors,20 patients with postoperative liver cancer,and 157 patients with benign liver disea-ses.The levels of GNB4 and Riplet gene methylation in plasma were detected using quantitative methylation-specific PCR(qMSP).Serum alpha-fetoprotein(AFP)levels were measured by direct chemiluminescence.Results The sensitivity and specificity of AFP in diagnosis were 51.3％and 94.3％,respectively;the sensitivity and specificity of GNB4 gene methylation in diagnosis were 83.3％and 99.4％,respectively;the sensitivity and specificity of Riplet gene methylation in diagnosis were 73.1％and 99.4％,respectively.The sensitivity and specificity of GNB4 and Riplet gene methylation combined diagnosis were 92.3％and 98.7％,respectively;the sensitivity and specificity of AFP,GNB4 and Riplet gene methylation combined diagnosis were 92.3％and 98.7％,respectively;the sensitivity and specificity of combined diagnosis including age and gender were 93.6％and 97.5％,respective-ly.Conclusion The sensitivity and specificity of AFP in the diagnosis of primary liver cancer are limited,while the methylation levels of GNB4 and Riplet genes are higher,and the sensitivity and specificity of their combined de-tection are higher than those of AFP.The sensitivity and specificity of AFP,GNB4 and Riplet gene methylation combined diagnosis are significantly higher than those of AFP,GNB4 and Riplet gene methylation alone.

OBJECTIVE To analyze the compositional differences between Fructus Tritici Levis and Triticum aestivum， and to provide reference for identification and quality control of both. METHODS Twenty batches of Fructus Tritici Levis and three batches of T. aestivum were collected， and their fingerprints were acquired by high-performance liquid chromatography and the similarities were evaluated by the Evaluation System of Similarity of Chromatographic Fingerprints of Traditional Chinese Medicine （2012 version）. Cluster analysis （CA）， principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA） were performed to analyze the difference of Fructus Tritici Levis and T. aestivum from different regions， and the differential components were screened. The contents of the six identified components in Fructus Tritici Levis and T. aestivum were determined. RESULTS The similarities of the fingerprints of Fructus Tritici Levis ranged from 0.928 to 0.996， and the relative similarities of T. aestivum with Fructus Tritici Levis ranged from 0.761 to 0.773. A total of 19 common peaks were calibrated， and six components including linolenic acid， linoleic acid， 5-heptadecylresorcinol， 5-nonadodecylresorcinol， 5- heneicosylresorcinol， and 5-tricosylresorcinol were identified. The results of CA and PCA showed that Fructus Tritici Levis and T. aestivum could be clearly distinguished； the distribution of Fructus Tritici Levis from Anhui province was relatively concentrated. The results of OPLS-DA showed that linolenic acid， linoleic acid， and other six unknown compounds were the differential components between Fructus Tritici Levis and T. aestivum. The average contents of the six identified components in Fructus Tritici Levis were 0.100 9， 1.094 0， 0.005 1， 0.030 9， 0.098 2，and 0.024 8 mg/g， respectively； the contents of linolenic acid and linoleic acid in Fructus Tritici Levis were significantly higher than those in T. aestivum （P＜0.05）.CONCLUSIONS The established qualitative and quantitative methods are simple and reliable， and can be used for the identification and quality evaluation of Fructus Tritici Levis and T. aestivum. The identified differential components， such as linolenic acid and linoleic acid， can also provide clues for the differentiation and pharmacological study of Fructus Tritici Levis and T. aestivum.