Aim To investigate the effect of vaccarin on mouse atherosclerosis in vivo and the underlying mechanism. Methods AopE mice aged 6 to 8 weeks old were used to establish the atherosclerosis model. Oil red O staining was used to determine the lipid levels in aorta and aortic root. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of serum inflammatory factors. Results Vaccarin could effectively reduce the levels of blood glucose and blood pressure in AopE

OBJECTIVE:To optimize the extraction technology of glucosinolates from Uighur medicine Brassica rapa L.. METHODS:Ethanol refluxing method was adopted to extract glucosinolates from Uighur medicine B. rapa L.. With the comprehen-sive score of glucosinolates and dry extract yield as the index,L9(34)orthogonal test based on the single factor test was adopted to observe the effects of ethanol volume fraction,extraction time and material-liquid ratio on the extraction technology of glucosino-lates from B. rapa L.,and verification test was conducted. RESULTS:The optimal extraction technology was to add 95% ethanol 8 times as much as the amount of the herbs,twice for 1.0 h reflux extractions. The extraction amount of glucosinolates and dry ex-tract yield were 7.36 mg/g and 25.29% at average,respectively.The comprehensive score of RSD was 0.52%(n=3). CONCLU-SIONS:The optimal extraction technology is stable and feasible and can be used for the extraction of glucosinolates from B. rapa L..

Background: Phototherapy is an important method to treatvitiligo. However, it is unclear how phototherapy affectsmelanocyte precursors and skin neural crest stem cells. Objective: To investigate the underlying mechanisms of narrow-band ultraviolet B (NB-UVB) induced melanocyte lineagedifferentiated from human scalp-derived neural creststem cells (HS-NCSCs). Methods: HS-NCSCs were expandedfrom scalp hair follicles. The c-Kit−/CD57− HS-NCSCs wereisolated by cell sorting. Different doses of NB-UVB wereused to irradiate these HS-NCSCs. Cell ultrastructure was examinedby transmission electron microscope. Melanocytemarker expression was analyzed by Quantitative RT-PCRand Western blot. Cell proliferation and migration were alsoevaluated. Results: The c-Kit−/CD57− HS-NCSCs expressedembryonic NCSC biomarkers. NB-UVB at a dose of 100 mJof NB-UVB had little effect on the cell proliferation of differentiatedmelanocytes from c-Kit−/CD57− HS-NCSCs, while700 mJ inhibited cell proliferation significantly. The dendriticprocesses of differentiated melanocytes increased afterradiation. The tyrosinase and Melanocortin 1 receptor (Mc1R)expression of differentiated melanocytes increased after NB-UVB exposure. The effect of NB-UVB on tyrosinase expressionwas modulated by signaling inhibitors H89 andPD98059 as well as Mc1R level in the cells. The migrationability of differentiated melanocytes was enhanced under100 mJ exposure. Conclusion These data demonstrate thatNB-UVB facilitates melanocytic differentiation of the HSNCSCsand enhances migration of these cells. Mc1R andcAMP pathway play a critical role in NB-UVB induced melanocyticdifferentiation.

Mitophagy is a process whereby cells selectively remove mitochondria through the mechanism of autophagy, which plays an important role in maintaining cellular homeostasis. In order to explore the effect of mitophagy genes on the antioxidant activities of Saccharomyces cerevisiae, mutants with deletion or overexpression of mitophagy genes ATG8, ATG11 and ATG32 were constructed respectively. The results indicated that overexpression of ATG8 and ATG11 genes significantly reduced the intracellular reactive oxygen species (ROS) content upon H₂O₂ stress for 6 h, which were 61.23% and 46.35% of the initial state, respectively. Notable, overexpression of ATG8 and ATG11 genes significantly increased the mitochondrial membrane potential (MMP) and ATP content, which were helpful to improve the antioxidant activities of the strains. On the other hand, deletion of ATG8, ATG11 and ATG32 caused mitochondrial damage and significantly decreased cell vitality, and caused the imbalance of intracellular ROS. The intracellular ROS content significantly increased to 174.27%, 128.68%, 200.92% of the initial state, respectively, upon H₂O₂ stress for 6 h. The results showed that ATG8, ATG11 and ATG32 might be potential targets for regulating the antioxidant properties of yeast, providing a new clue for further research.
Mitophagy/genetics* ; Saccharomyces cerevisiae/genetics* ; Antioxidants ; Hydrogen Peroxide/pharmacology* ; Reactive Oxygen Species

As a branched chain amino acid, L-valine is widely used in the medicine and feed sectors. In this study, a microbial cell factory for efficient production of L-valine was constructed by combining various metabolic engineering strategies. First, precursor supply for L-valine biosynthesis was enhanced by strengthening the glycolysis pathway and weakening the metabolic pathway of by-products. Subsequently, the key enzyme in the L-valine synthesis pathway, acetylhydroxylate synthase, was engineered by site-directed mutation to relieve the feedback inhibition of the engineered strain. Moreover, promoter engineering was used to optimize the gene expression level of key enzymes in L-valine biosynthetic pathway. Furthermore, cofactor engineering was adopted to change the cofactor preference of acetohydroxyacid isomeroreductase and branched-chain amino acid aminotransferase from NADPH to NADH. The engineered strain C. glutamicum K020 showed a significant increase in L-valine titer, yield and productivity in 5 L fed-batch bioreactor, up to 110 g/L, 0.51 g/g and 2.29 g/(L‧h), respectively.
Valine ; Corynebacterium glutamicum/genetics* ; Metabolic Engineering ; Amino Acids, Branched-Chain ; Bioreactors

As self-subunit swapping chaperones or metallochaperones, the activators assist nitrile hydratases to take up metal ions and they are essential for active expression of nitrile hydratases. Compared with nitrile hydratases, the activators have a low sequence identity. Study of the activation characteristics and the relationships between structures and functions of the activators is of great significance for understanding the maturation mechanism of nitrile hydratase. We co-expressed low-molecular-mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1 with four heterologous activators respectively and determined their activation abilities. Then we made sequence analysis and structure modelling, and studied the functions of the important domains of the activators. Results showed that all four heterologous activators could activate L-NHase, however, the specific activities of L-NHases were different after activation. L-NHase showed the highest specific activity after being activated by activator A, which was 97.79% of that of the original enzyme, but the specific activity of L-NHase after being activated by activator G was only 23.94% of that of the original enzyme. Activator E and activator G had conserved domains (TIGR03889), and deletion of their partial sequences resulted in a substantial loss of activation abilities for both activators. Replacing the N-terminal sequence of activator G with the N-terminal sequence of activator E, and adding the C-terminal sequence of activator E to the C-terminus of activator G could increase the specific activity of L-NHase by 178.40%. The activation by nitrile hydratase activators was universal and specific, and the conserved domains of activators were critical for activation, while the N-terminal domain and C-terminal domain also had important effects on activation.

The zearalenone hydrolase (ZHD101) derived from Clonostachys rosea can effectively degrade the mycotoxin zearalenone (ZEN) present in grain by-products and feed. However, the low thermal stability of ZHD101 hampers its applications. High throughput screening of variants using spectrophotometer is challenging because the reaction of hydrolyzing ZEN does not change absorbance. In this study, we used ZHD101 as a model enzyme to perform computation-aided design followed by experimental verification. By comparing the molecular dynamics simulation trajectories of ZHD101 at different temperatures, 32 flexible sites were selected. 608 saturated mutations were introduced into the 32 flexible sites virtually, from which 12 virtual mutants were screened according to the position specific score and enzyme conformation free energy calculation. Three of the mutants N156F, S194T and T259F showed an increase in thermal melting temperature (ΔTm>4 °C), and their enzyme activities were similar to or even higher than that of the wild type (relative enzyme activity 95.8%, 131.6% and 169.0%, respectively). Molecular dynamics simulation analysis showed that the possible mechanisms leading to the improved thermal stability were NH-π force, salt bridge rearrangement, and hole filling on the molecular surface. The three mutants were combined iteratively, and the combination of N156F/S194T showed the highest thermal stability (ΔTm=6.7 °C). This work demonstrated the feasibility of engineering the flexible region to improve enzyme performance by combining virtual computational mutations with experimental verification.
Computer-Aided Design ; Edible Grain ; Enzyme Stability ; Hydrolases/metabolism* ; Hypocreales/enzymology* ; Protein Engineering ; Zearalenone

Proteus mirabilis lipase (PML) features tolerance to organic solvents and great potential for biodiesel synthesis. However, the thermal stability of the enzyme needs to be improved before it can be used industrially. Various computational design strategies are emerging methods for the modification of enzyme thermal stability. In this paper, the complementary algorithm-based ABACUS, PROSS, and FoldX were employed for positive selection of PML mutations, and their pairwise intersections were further subjected to negative selection by PSSM and G_REMLIN to narrow the mutation library. Thereby, 18 potential single-point mutants were screened out. According to experimental verification, 7 mutants had melting temperature (T_m) improved, and the ΔT_m of K208G and G206D was the highest, which was 3.75 ℃ and 3.21 ℃, respectively. Five mutants with activity higher than the wild type (WT) were selected for combination by greedy accumulation. Finally, the T_m of the five-point combination mutant M10 increased by 10.63 ℃, and the relative activity was 140% that of the WT. K208G and G206D exhibited certain epistasis during the combination, which made a major contribution to the improvement of the thermal stability of M10. Molecular dynamics simulation indicated that new forces were generated at and around the mutation sites, and the rearrangement of forces near G206D/K208G might stabilize the Ca²⁺ binding site which played a key role in the stabilization of PML. This study provides an efficient and user-friendly computational design scheme for the thermal stability modification of natural enzymes and lays a foundation for the modification of PML and the expansion of its industrial applications.
Enzyme Stability ; Lipase/chemistry* ; Molecular Dynamics Simulation ; Proteus mirabilis/metabolism* ; Solvents/chemistry*

OBJECTIVE: To explore clinical effect of repairing anterior talofibular ligament with knot-free anchors under total ankle arthroscopy in treating chronic lateral ankle instability. METHODS: From April 2018 to August 2021, 24 patients with chronic lateral ankle instability were treated with knot-free anchors under total ankle arthroscopy to repair anterior talofibular ligament, including 16 males and 8 females, aged from 22 to 42 years old with an average of(28.6±5.8) years old;the time from injury to opertaion ranged from 6 to 10 months with an average of(7.7±1.3) months. Preoperative and postoperative American Orhopaedic Foot and Ankle Society (AOFAS) score, visual analogue scale (VAS), talar tilt, anterior talar translation(ATT) were recorded and compared. RESULTS: All patients were followed up from 10 to 12 months with an average of (10.2±1.14) months. Incision were healed at stageⅠ, and no infection, nerve injury and lateral ankle instability occurred. AOFAS score improved from(52.79±8.96) before opertaion to (93.00± 4.01) at 6 months after operation, 23 patients got excellent result and 1 good;VAS decreased from (5.50±0.98) before opertaion to (1.04±0.80) at 6 months after operation(P<0.05);talar tilt decreased from(9.16±2.09)° to (3.10±1.72)° at 3 months after operation(P<0.05);ATT decreased from(8.80±2.55) mm to (2.98±1.97) mm at 3 months after operation(P<0.05). Twenty-four patients drawer test and varus-valgus rotation wer negative. CONCLUSION Repairing anterior talofibular ligament with knot-free anchors under total ankle arthroscopy for the treatment of chronic lateral ankle instability has advantages of less trauma, less complications safe and reliable, and good recovery of ankle joint function.
Female ; Male ; Humans ; Young Adult ; Adult ; Ankle Joint/surgery* ; Ankle ; Arthroscopy ; Lateral Ligament, Ankle/surgery* ; Joint Instability/surgery*

Chemotherapy is one of the main options in clinical tumor treatment. Although chemotherapy drugs have a good therapeutic effect, they can also cause a series of adverse reactions, such as neurotoxicity. Chemotherapy-induced neurotoxicity is a dose-limi-ting adverse reaction that significantly affects patients' long-term treatment and quality of life. This article reviewed literature from 2000 to the present on chemotherapy-induced neurotoxicity and found that oxaliplatin was the most frequently used chemotherapy drug. Based on the clinical characteristics of oxaliplatin-induced neurotoxicity, this article summarized the understanding of its pathogenesis from both traditional Chinese medicine(TCM) and western medicine perspectives, discussed the role and mechanism of TCM compounds and monomeric components, and explored the research direction of using cutting-edge biotechnology to reveal the mechanism of oxaliplatin-induced neurotoxicity from a temporal-spatial perspective of intercellular communication and the application prospects of an interdisciplinary model combining TCM pathogenesis, western medicine manifestations, and artificial intelligence in precise intervention decision-making for TCM, aiming to provide research ideas for the prevention and treatment of oxaliplatin-induced neurotoxicity and the development of new drugs.
Humans ; Medicine, Chinese Traditional ; Oxaliplatin/adverse effects* ; Artificial Intelligence ; Quality of Life ; Drugs, Chinese Herbal/therapeutic use* ; Antineoplastic Agents/adverse effects* ; Cognition