Objective To observe the protective effect of electroacupuncture(EA)on ankle joint synovitis and HIF-1 α/MDM2/p53 signaling pathway in adjuvant arthritis(AA)rats.Methods SD rats were randomly divided into control group,model group,sham EA group,agonist group,EA group,EA+antagonist group,antagonist group,with 10 rats in each group.Preparation of AA rat model by injecting Freund's complete adjuvant into bilateral foot pads of rats.EA(2 Hz,3 V)was applied to bilateral ST36 and GB39 or two control points(5 mm left to ST36 and GB39)for 15 min,once every other day for a total of 8 times.HIF-1 α agonists and antagonists were injected into the tail vein of rats in the corresponding groups.The inflammatory conditions of the ankle joint were observed by HE staining,and the contents of serum HIF-1 α was assayed by ELISA.mRNA expression of HIF-1α,VEGF,MDM2 and p53 were detected by RT-PCR,protein expression of HIF-1α,VEGF,MDM2 and p53 were detected by Western blot in synovium of AA rats.Results The results of HE staining showed that compared with the control group,the synovial cells in the model group had significant proliferation and inflammatory cell infiltration(P<0.01);Compared with the model group,the arthritic reaction of the rats in the EA group,the EA+antagonist group and the antagonist group was significantly reduced(P<0.01),while the arthritic reaction of the rats in the sham EA group and the agonist group was still serious(P>0.05).The results of ELISA showed that compared with the control group,the serum HIF-1αexpression of the model group rats was significantly increased(P<0.01);Compared with model group,serum HIF-1αexpression of rats in EA group,EA+ antagonist group and antagonist group was significantly decreased(P<0.01);There was no significant difference in serum HIF-1αexpression among the model group,sham EA group and agonist group(P>0.05).RT-PCR results showed that compared with the control group,the mRNA expression of HIF-1α,VEGF,MDM2 and p53 of model group rats increased significantly;Compared with model group,the mRNA expression of HIF-1α,VEGF and MDM2 of rats in EA group,EA+antagonist group and antagonist group decreased significantly,while the mRNA expression of p53 increased significantly.Western blot results showed that compared with the control group,the protein expression of HIF-1α,VEGF,MDM2 and p53 of model group rats increased significantly;Compared with model group,the mRNA expression of HIF-1α,VEGF and MDM2 of rats in EA group,EA+antagonist group and antagonist group decreased significantly,while the mRNA expression of p53 increased significantly.Conclusion Electroacupuncture intervention has significant protective effect on ankle inflammation in AA rats,HIF-1 α/MDM2/p53 signaling pathway plays a key role in this process.

Yeast autolysis affects the flavor and quality of beer. The regulation of yeast autolysis is a need for industrial beer production. Previous studies on brewer's yeast autolysis showed that the citric acid cycle-related genes had a great influence on yeast autolysis. To explore the contribution of isocitrate dehydrogenase genes in autolysis, the IDP1 and IDP2 genes were destroyed or overexpressed in typical lager yeast Pilsner. The destruction of IDP1 gene improved the anti-autolytic ability of yeast, and the anti-autolytic index after 96 h autolysis was 8.40, 1.5 times higher than that of the original strain. The destruction of IDP1 gene increased the supply of nicotinamide adenine dinucleotide phosphate (NADPH) and the NADPH/NADP⁺ ratio was 1.94. After fermentation, intracellular ATP level was 1.8 times higher than that of the original strain, while reactive oxygen species (ROS) was reduced by 10%. The destruction of IDP2 gene resulted in rapid autolysis and a decrease in the supply of NADPH. Anti-autolytic index after 96 h autolysis was 4.03 and the NADPH/NADP⁺ ratio was 0.89. After fermentation, intracellular ATP level was reduced by 8% compared with original strain, ROS was 1.3 times higher than that of the original strain. The results may help understand the regulation mechanism of citric acid cycle-related genes on yeast autolysis and provide a basis for the selection of excellent yeast with controllable anti-autolytic performance.
Humans ; Isocitrate Dehydrogenase/genetics* ; NADP ; Reactive Oxygen Species ; Autolysis ; Adenosine Triphosphate

OBJECTIVE: To investigate the coexisting mutations and clinical significance of Homo sapiens neuroblastoma RAS viral oncogene homolog (NRAS) gene in acute myeloid leukemia (AML) patients. METHODS: High-throughput DNA sequencing and Sanger sequencing were used to detect 51 gene mutations. The occurrence, clinical characteristics and treatment efficacy of coexisting genes with NRAS were investigated. RESULTS: A total of 57 NRAS mutations (17.5%) were detected in 326 patients with AML. Compared with the patients in NRAS non-mutation group, patients in the mutant group were younger (P=0.018) and showed lower platelet count (P=0.033), but there was no significant difference in peripheral leukocyte count, hemoglobin, and sex. For FAB classification, NRAS mutation and M2 subtype showed mutually exclusive (P=0.038). Among 57 patients carried with NRAS mutation, 51 (89.5%) patients carried with other gene mutations, 25 (43.9%) carried with double gene mutations, 10 (17.5%) carried with 3 gene mutations, and 16 (28.1%) corried with ≥ 4 gene mutations. The most common coexisting gene mutation was KRAS (24.6%, 14/57), followed by FLT3-ITD (14.0%, 8/57), RUNX1 (12.3%, 7/57), NPM1 (10.5%, 6/57), PTPN11 (10.5%, 6/57), DNMT3A (10.5%, 6/57) and so on. The age (P=0.013, P=0.005) and peripheral platelet count (P=0.007, P=0.021) of patients with NPM1 or DNMT3A mutations were higher than those of the patients with wild type, but there was no significant difference in peripheral leukocyte count and hemoglobin. Also, there was no significant difference in age, peripheral leukocyte count, hemoglobin, and peripheral platelet count between the patients in KRAS, FLT3-ITD, RUNX1 or PTPN11 mutant group and the wild group. Patients with FLT3-ITD mutations showed a lower complete remission (CR) rate (P=0.044). However, there was no significant difference in CR rate between the patients with KRAS, NPM1, RUNX1, PTPN11 or DNMT3A mutations and the wild group. The CR rate of the patents with single gene mutation, double gene mutations, 3 gene mutations, and≥ 4 gene mutations were decreased gradually, and there was no significant difference in CR rate between pairwise comparisons. CONCLUSION The mutation rate of NRAS mutation is 17.5%, 89.5% of AML patients with NRAS mutation coexist with additional gene mutations. The type of coexisting mutations has a certain impact on clinical characteristics and CR rate of patients with AML.
Core Binding Factor Alpha 2 Subunit/genetics* ; GTP Phosphohydrolases/genetics* ; Humans ; Leukemia, Myeloid, Acute/genetics* ; Membrane Proteins/genetics* ; Mutation ; Nucleophosmin ; Prognosis ; Proto-Oncogene Proteins p21(ras)/genetics* ; fms-Like Tyrosine Kinase 3

Objective To investigate the effect of phenformin combined with hexokinase inhibitor 2-deoxyglucose (2-DG) on the treatment of triple-negative breast cancer cell lines 4T1 and MDA-MB-231. Methods Following treatment with phenformin, 2-DG or phenformin combined with 2-DG on 4T1 and MDA-MB-231 cells for 48 h, the cell proliferation in each group was detected by SRB and the apoptosis of cells was detected by flow cytometry. The concentration of glucose and lactic acid in cell culture supernatant was detected by ELISA. The activity of mitochondrial respiratory chain complex Ⅰ was detected by FlexStation3 and the mitochondrial oxygen consumption (OCR) was assayed with the Seahorse X Fe Analyzer. Results The hexokinase expression (4.6±0.17,3.73±0.21), glucose consumption (356±31，397±42) μg/10⁵ cells , Lactic acid concentration (5.59±0.52, 7.83±0.78) μmol/L in the supernatant of 4T1 and MDA-MB-231 cells in Phenformin group were higher than that in control group ( 1±0.15，1±0.12 ) , ( 289±25，301±32) μg/10⁵cells , ( 2.37±0.18，4.01±0.45) μmol/L (P < 0.01). Even if the dose was reduced by 90%, the cell viability of phenformin combined with 2-DG group (64.63±2.28, 51.97±2.29) % was still higher than that of phenformin group (86.70±1.83, 85.53±1.46) % (P<0.001). The combination of the two drugs significantly promoted the apoptosis of 4T1 and MDA-MB-231. In addition, compared with the phenformin group (5.59±0.52, 7.83±0.78) μmol/L, the phenformin combined with 2-DG group (3.46±0.37, 5.18±0.62) μmol/L cell lactic acid production also greatly reduced (P<0.01). Compared with the phenformin or 2-DG single-drug group, the phenformin combined with 2-DG group can significantly inhibit the growth rate of tumors in tumor-bearing mice (P<0.01). The median survival time of tumor-bearing mice in the phenformin combined with 2-DG group was 72.5 d, which was higher than that in the phenformin group 57 d and 2-DG group 55.5 d (P<0.01). Conclusion Hexokinase inhibitor 2-DG significantly enhances the therapeutic effects of phenformin on triple-negative breast cancer cells.

Genistein and its monoglucoside derivatives play important roles in food and pharmaceuticals fields, whereas their applications are limited by the low water solubility. Glycosylation is regarded as one of the effective approaches to improve water solubility. In this paper, the glycosylation of sophoricoside (genistein monoglucoside) was investigated using a cyclodextrin glucosyltransferase from Penibacillus macerans (PmCGTase). Saturation mutagenesis of D182 from PmCGTase was carried out. Compared with the wild-type (WT), the variant D182C showed a 13.42% higher conversion ratio. Moreover, the main products sophoricoside monoglucoside, sophoricoside diglucoside, and sophoricoside triglucoside of the variant D182C increased by 39.35%, 56.05% and 64.81% compared with that of the WT, respectively. Enzymatic characterization showed that the enzyme activities (cyclization, hydrolysis, disproportionation) of the variant D182C were higher than that of the WT, and the optimal pH and temperature of the variant D182C were 6 and 40℃, respectively. Kinetics analysis showed the variant D182C has a lower K_m value and a higher k_cat/K_m value than that of the WT, indicating the variant D182C has enhanced affinity to substrate. Structure modeling and docking analysis demonstrated that the improved glycosylation efficiency of the variant D182C may be attributed to the increased interactions between residues and substrate.
Cyclodextrins ; Genistein ; Glucosyltransferases/metabolism* ; Glycosylation ; Kinetics

OBJECTIVE: To investigate the diagnostic value of multislice spiral CT (MSCT) and MRI in occult fracture of knee joint with meniscus and ligament injury. METHODS: From January 2020 to March 2021, 63 patients with knee occult fracture with meniscus and ligament injury, including 41 males and 22 females, aged from 21 to 67 years old, with an average of (44.35±8.77) years old, the course of disease ranged from 1 to 6 days, with an average of (4.64±1.75) days, the body mass index (BMI) was (19.85±2.78) kg/m². MSCT and MRI data of 63 patients were collected and statistically analyzed to evaluage their diagnist value. RESULTS: The detection of MRI for occult knee fravtures with meniscus and ligament injury, joint cavity effusion, bone marrow edema, and articular surface injury were 100.00% (63/63), 95.24% (60/63), 42.86% (27/63) and 36.51% (23/63), respectively. The detection rates of MSCT were 49.21% (31/63), 41.27% (26/63), 0.00% (0/63) and 1.59% (1/63), respectively, significantly lwver than that of MRI (P<0.05). The diagnostic sensitivity, specificity and accuracy of MRI were significantly higher than those of MSCT(P<0.05). CONCLUSION The sensitivity, specificity and accuracy of magnetic resonance imaging in the diagnosis of occult fracture of knee joint with meniscus and ligament injury are significantly better than that of MSCT. MRI has higher accuracy in the diagnosis of peripheral tissue diseases such as joint cavity, articular surface and bone marrow, and can reduce the risk of clinical misdiagnosis.
Male ; Female ; Humans ; Young Adult ; Adult ; Middle Aged ; Aged ; Tibial Meniscus Injuries/diagnostic imaging* ; Fractures, Closed/diagnostic imaging* ; Arthroscopy/methods* ; Knee Injuries/diagnostic imaging* ; Knee Joint/diagnostic imaging* ; Magnetic Resonance Imaging/methods* ; Ligaments ; Meniscus ; Tomography, Spiral Computed ; Fractures, Bone ; Anterior Cruciate Ligament Injuries

To quickly and efficiently understand the intracellular metabolic characteristics of industrial microorganisms, and to find potential metabolic engineering targets, genome-scale metabolic network models (GSMMs) as a systems biology tool, are attracting more and more attention. We review here the 20-year history of metabolic network model, analyze the research status and development of GSMMs, summarize the methods for model construction and analysis, and emphasize the applications of metabolic network model for analyzing intracellular metabolic activity of microorganisms from cellular phenotypes, and metabolic engineering. Furthermore, we indicate future development trend of metabolic network model.
Industrial Microbiology ; Metabolic Engineering ; Metabolic Networks and Pathways ; genetics ; Models, Biological ; Systems Biology

OBJECTIVE: To investigate clinical results of reverse total shoulder arthroplasty in treating old fracture of proximal humerus in elderly patients. METHODS: From January 2012 to December 2017, 12 elderly patients with old proximal humeral fractures were treated with reverse total shoulder arthroplasty. There were 5 males and 7 females with an average age of 70.2 years old (ranged from 63 to 81 years old) and an average course of 12.3 months (ranged from 9 to 18 months). VAS score was used to evaluate the degree of pain relief of shoulder joint, Constant-Murley score was used to evaluate the improvement of shoulder joint function and observe the complications during and after operation. RESULTS: All the 12 patients were followed up with an average duration of 22.3 months (ranged from 15 to 56 months). At the latest follow-up, the VAS score was 1.9±1.4, and Constant-Murley score was 83.4±8.4. Among them, shoulder joint flexion was (92.5±7.7)°, abduction was (90.4±14.3)°, external rotation was(31.9±10.0)°, and internal rotation was(58.9±13.1)°. There was statistical significance before and after treatment(<0.05). There were 2 cases with glenoid notch. The imaging findings were grade I and grade II respectively. No loosening was found during follow-up. No infection, acromial fracture, prosthesis loosening, axillary nerve injury and other complications occurred. CONCLUSIONS Reverse total shoulder arthroplasty in treating old proximal humeral fractures in elderly patients can achieve satisfactory results, however, indications and complications should be noted.
Aged ; Aged, 80 and over ; Arthroplasty, Replacement, Shoulder ; Female ; Humans ; Humerus ; Male ; Middle Aged ; Shoulder ; Shoulder Fractures ; surgery ; Shoulder Joint ; Treatment Outcome

OBJECTIVE: To set up an easy and effective method for biotinylation of small molecule drugs with long chain. METHODS: Biotinylated 6-aminocaproic acid was synthesized as intermediate by one step method, doxorubicin(DOX) with auto-fluorescence was used as the first drug, and by DCC and DMAP catalysis, biotinylated DOX was synthesized. Using the double fluorescence system of DOX, the binding ability of biotinylated DOX to avidin and its biological activity were determined. When verified to be reasonable and effective, the method was applied to catalyze biotinylated paclitexal (PTX) which didn′t have auto-fluorescence itself, and the physical and chemical characteristics, and biological activities as well as the visualization were tested. RESULTS: The binding rate of synthesized DOX to avidin was 93.7%; the cells inhibition rate and localization were the same as DOX; the purity of biotinylated PTX was 84.42%, and the structure shown by NMR was correct; the cell inhibition rate was the same as PTX; the combination of PTX with microtubules was observed by visual modification. CONCLUSION: The method supplies a temperate way for biotinylation, and can be used for the synthesis and visualization of small molecules as probes and research of drug mechanism.

Glucaric acid (GA), a top value-added chemical from biomass, has been widely used for prevention and control of diseases and the production of polymer materials. In GA biosynthesis pathway, the conversion of inositol to glucuronic acid that catalyzed by myo-inositol oxygenase is the limiting step. It is necessary to improve MIOX activity. In the present study, we constructed a high-throughput screening system through combing the concentration of GA with the green fluorescent protein fluorescence intensity. By applying this screening system, three positive variants (K59V/R60A, R171S and D276A) screened from the mutant library. In comparison, the recombinant strain Escherichia coli BL21(DE3)/MU-R171S accumulated more GA, 136.5% of that of the parent strain.
Biosensing Techniques ; Biosynthetic Pathways ; Escherichia coli ; Glucaric Acid ; chemistry ; Inositol Oxygenase ; chemistry