Diacylglycerol (DAG) is an intermediate product in lipid metabolism and plays an important physiological role in human body. It is mainly prepared by hydrolyzing lipid with lipase. However, research on the detection method of 1, 2-diacylglycerol (1, 2-DAG) and 1, 3-diacylglycerol (1, 3-DAG) and catalytic specificity of lipase was not enough, which limits its wide application. To address these challenges, an efficient quantitative detection method was first established for 1, 2-DAG (0.025-0.200 g/L) and 1, 3-DAG (0.025-0.150 g/L) by combining supercritical fluid chromatography with evaporative light scattering detector and optimizing the detection and analysis parameters. Based on the molecular docking between Thermomyces lanuginosus lipase (TLL) and triolein, five potential substrate binding sites were selected for site-specific saturation mutation to construct a mutation library for enzyme activity and position specificity screening. The specificity of sn-1, 3 of the I202V mutant was the highest in the library, which was 11.7% higher than the specificity of the wild type TLL. In summary, the position specificity of TLL was modified based on a semi-rational design, and an efficient separation and detection method of DAG isomers was also established, which provided a reference for the study of the catalytic specificity of lipase.
Humans ; Diglycerides ; Molecular Docking Simulation ; Binding Sites ; Catalysis ; Lipase/genetics*

Shikimic acid (SA), as a hydroaromatic intermediate in the common pathway of aromatic amino acid biosynthesis, is the starting material for the synthesis of neuraminidase inhibitors and other useful compounds. The fermentative production of SA by metabolically engineered microorganisms is an excellent alternative to the extraction from fruits of the Illicium plant. In this study, Escherichia coli was metabolically engineered by rational design and genetic manipulation for fermentative production of SA. Firstly, blocking the aromatic amino acid pathway after the production of SA was carried out by deletion of aroL and aroK genes encoding SA kinase. Secondly, the ptsG gene encoding protein EIICBglc were removed in the aroL/aroK mutant strain to make the phosphotransferase system (PTS) system default. In the resulting strain, the phosphoenolpyruvate-dependent PTS pathway, a main pathway for glucose transport, were replaced by ATP-dependent GalP (galactose permease). Thus, more PEP flux was used to produce SA as a critical precursor of SA. Furthermore, ydiB gene (encoding quinic acid/SA dehydrogenase) was deleted to prevent SA precursors of 3-dehyroquinic acid into the byproduct of quinic acid. Thus, the engineered strain with four genes deletion was constructed and 576 mg/L SA was produced in the shake flask fermentation. Results show that SA produciton was increased 90 times compared to the parent strain E. coli CICIM B0013.
Escherichia coli ; enzymology ; genetics ; metabolism ; Gene Knockout Techniques ; Metabolic Engineering ; methods ; Recombinant Proteins ; genetics ; metabolism ; Shikimic Acid ; metabolism

OBJECTIVE:To provide reference for exploring the strategy of promoting TCM formula granules.METHODS:The questionnaire was designed to investigate the cognition and attitude of TCM practitioners from different grades to TCM formula granules in our university,and the investigation data were analyzed statistically.RESULTS:A total of 312 questionnaires were collected and 306 questionnaires were collected with recovery rate of 98.08％.Among them,305 questionnaires were effective with effective rate of 99.67％.46.89％ of interviewed practitioners were found to know and understand about TCM formula granule,52.46％ knew but did not understand;the higher the grade,the higher the proportion of the known and the better understanding (P＜0.05);the cognitive channels included diagnosis and treatment course (69.64％),took by others (65.02％),took by oneself (51.49％).TCM formula granules were easy to take without decoction (95.71％) and easy to carry (87.13％),the advantage of which had high popularity,but there was not much knowledge about other advantages.7.54％ of interviewed practitioners fully appreciated TCM formula granules and 69.18％ recognized them;the higher the grade,the higher the degree of recognition (P＜ 0.05).89.51％ were willing to learn more about TCM formula granules.60.98％ said they would prescribed TCM formula granules in the furure.Among the influential factors for the attitude of the practitioners to TCM formula granules,the three factors with the highest influence were the influence of the teachers,the influence of the patients and their own practice.CONCLUSIONS:The practice of TCM practitioners in our university in Beijing is not ideal for the cognition of TCM formula granules.There is still a great room for improvement in the attitude to TCM formula granules.The teachers and patients are key influential factors for the attitude to TCM formula granules.

Sialyllactose is one of the most abundant sialylated oligosaccharides in human milk oligosaccharides (HMOs), which plays an important role in the healthy development of infants and young children. However, its efficient and cheap production technology is still lacking presently. This study developed a two-step process employing multiple-strains for the production of sialyllactose. In the first step, two engineered strains, E. coli JM109(DE3)/ pET28a-BT0453 and JM109(DE3)/pET28a-nanA, were constructed to synthesize the intermediate N-acetylneuraminic acid. When the ratio of the biomass of the two engineered strains was 1:1 and the reaction time was 32 hours, the maximum yield of N-acetylneuraminic acid was 20.4 g/L. In the second step, E. coli JM109(DE3)/ pET28a-neuA, JM109(DE3)/ pET28a-nst and Baker's yeast were added to the above fermentation broth to synthesize 3'-sialyllactose (3'-SL). Using optimal conditions including 200 mmol/L N-acetyl-glucosamine and lactose, 150 g/L Baker's yeast, 20 mmol/L Mg²⁺, the maximum yield of 3'-SL in the fermentation broth reached 55.04 g/L after 24 hours of fermentation and the conversion rate of the substrate N-acetyl-glucosamine was 43.47%. This research provides an alternative technical route for economical production of 3'-SL.
Child ; Humans ; Child, Preschool ; N-Acetylneuraminic Acid ; Escherichia coli/genetics* ; Lactose ; Fermentation ; Saccharomyces cerevisiae ; Oligosaccharides ; Glucosamine

Members of genus Acanthamoeba are widely distributed in the environment. Some are pathogenic and cause keratitis and fatal granulomatous amoebic encephalitis. In this study, we isolated an Acanthamoeba CJW/W1 strain from tap water in Wuxi, Jiangsu Province, China. Its 18S rDNA was sequenced and a phylogenetic tree was constructed. The isolated cysts belonged to morphologic group II. Comparison of 18S rDNA sequences of CJW/W1 strain and other isolates showed high similarity (99.7%) to a clinical isolate Asp, KA/E28. A phylogeny analysis confirmed this isolate belonged to the pathogenic genotype T4, the most common strain associated with Acanthamoeba-related diseases. This is the first report of an Acanthamoeba strain isolated from tap water in Wuxi, China. Acanthamoeba could be a public health threat to the contact lens wearers and, therefore, its prevalence should be monitored.
Acanthamoeba ; China ; DNA, Ribosomal ; Encephalitis ; Genotype ; Keratitis ; Phylogeny ; Prevalence ; Public Health ; Trees ; Viperidae ; Water

Objective: To investigate the natural regression and related factors of high-grade squamous intraepithelial lesion (HSIL) in the cervix of childbearing age women, and to evaluate the applicability of conservative management for future fertility needs. Methods: This study included 275 patients of reproductive age with fertility needs, who were diagnosed as HSIL by biopsy from April 30, 2015 to April 30, 2022, including 229 cases (83.3%) cervical intraepithelial neoplasia (CIN) Ⅱ and 46 cases (16.7%) CIN Ⅱ-Ⅲ. They were followed-up without immediate surgery in the First Affiliated Hospital of Nanjing Medical University. The median follow-up time was 12 months (range: 3-66 months). The regression, persistence and progression of lesions in patients with HSIL were analyzed during the follow-up period, the influencing factors related to regression and the time of regression were analyzed. Results: (1) Of the 275 HSIL patients, 213 cases (77.5%, 213/275) experienced regression of the lesion during the follow-up period. In 229 CIN Ⅱ patients, 180 cases (78.6%) regressed, 21 cases (9.2%) persisted, and 28 cases (12.2%) progressed. In 46 CIN Ⅱ-Ⅲ patients, 33 cases (71.7%) regressed, 12 cases (26.1%) persisted, and 1 case (2.2%) progressed to invasive squamous cell carcinoma stage Ⅰ a1. There was no significant difference in the regression rate between the two groups (χ²=1.03, P=0.309). (2) The average age at diagnosis, age <25 years old at diagnosis were independent influencing factor of HSIL regression in univariate analysis (all P<0.05). There was no significant difference between HSIL regression and pathological grading, the severity of screening results, human papillomavirus (HPV) genotype, colposcopy image characteristics, number of biopsies during follow-up and pregnancy experience (all P>0.05). (3) The median regression times for patients aged ≥25 years and <25 years at diagnosis were 15 and 12 months, respectively. Kaplan-Meier analysis showed that age ≥25 years at diagnosis significantly increased the median regression time compared to <25 years (χ²=6.02, P=0.014). Conclusions: For HSIL patients of childbearing age, conservative management without immediate surgical intervention is preferred if CINⅡ is fully evaluated through colposcopy examination. Age ≥25 years at diagnosis is a risk factor affecting the prognosis of HSIL patients.
Pregnancy ; Humans ; Female ; Adult ; Cervix Uteri/pathology* ; Uterine Cervical Neoplasms/pathology* ; Uterine Cervical Dysplasia/pathology* ; Biopsy ; Colposcopy/methods* ; Squamous Intraepithelial Lesions/pathology* ; Carcinoma in Situ/pathology* ; Papillomaviridae/genetics* ; Papillomavirus Infections/diagnosis* ; Squamous Intraepithelial Lesions of the Cervix/pathology*

Antimalarials ; pharmacology ; Artemisinins ; pharmacology ; Humans ; Medicine, Chinese Traditional

Objective: To investigate reciprocal regulation between Fur and two RyhB homologs in Methods: Regulatory relationships were assessed by a combination of colony morphology assay, primer extension, electrophoretic mobility shift assay and DNase I footprinting. Results: Fur bound to the promoter-proximal DNA regions of Conclusion Fur and the two RyhB homologs exert negative reciprocal regulation, and RyhB homologs have a positive regulatory effect on biofilm formation in
Bacterial Proteins/metabolism* ; Biofilms ; Gene Expression Regulation, Bacterial/physiology* ; Yersinia pestis/physiology*