AIM:To investigate the effect of dyes,Remazol BrOrange yellow(RBY)and erythrosine(ERY),on the outcomes of immunoblotting analysis when used for staining the concentrate gel in sodium dodecyl sulfate-polyacryl-amide gel electrophoresis(SDS-PAGE).METHODS:Polyacrylamide gels were divided into five groups:the control group(prepared according to the conventional kit protocol),the RBY-stained group with a final concentration of 0.08 g/L,the RBY-stained group with a final concentration of 0.16 g/L,the ERY-stained group with a final concentration of 0.08 g/L,and the ERY-stained group with a final concentration of 0.8 g/L.Gels were prepared and subjected to electro-phoresis,followed by coomassie brilliant blue staining to visualize protein bands.Subsequently,proteins were transferred to PVDF membranes,which were then blocked,incubated with primary and secondary antibodies,washed,and finally ex-posed for imaging to observe the target protein vinculin bands.RESULTS:Compared with the unstained concentrate gel,the loading wells of the RBY or ERY pre-stained concentrate gel were more clearly visible.Analysis of the gels stained with coomassie brilliant blue after electrophoresis and marker visualization showed no significant different in protein elec-trophoretic mobility between prestained and unstained gels.Comparative analysis of the immunoblotting also indicated that the detection of protein samples transferred to PVDF membranes was unaffected.CONCLUSION:Prestaining concen-trate gels with RBY or ERY can enhance the efficiency of gel-based electrophoresis and immunoblotting analysis.

AIM:To investigate the effect of dyes,Remazol BrOrange yellow(RBY)and erythrosine(ERY),on the outcomes of immunoblotting analysis when used for staining the concentrate gel in sodium dodecyl sulfate-polyacryl-amide gel electrophoresis(SDS-PAGE).METHODS:Polyacrylamide gels were divided into five groups:the control group(prepared according to the conventional kit protocol),the RBY-stained group with a final concentration of 0.08 g/L,the RBY-stained group with a final concentration of 0.16 g/L,the ERY-stained group with a final concentration of 0.08 g/L,and the ERY-stained group with a final concentration of 0.8 g/L.Gels were prepared and subjected to electro-phoresis,followed by coomassie brilliant blue staining to visualize protein bands.Subsequently,proteins were transferred to PVDF membranes,which were then blocked,incubated with primary and secondary antibodies,washed,and finally ex-posed for imaging to observe the target protein vinculin bands.RESULTS:Compared with the unstained concentrate gel,the loading wells of the RBY or ERY pre-stained concentrate gel were more clearly visible.Analysis of the gels stained with coomassie brilliant blue after electrophoresis and marker visualization showed no significant different in protein elec-trophoretic mobility between prestained and unstained gels.Comparative analysis of the immunoblotting also indicated that the detection of protein samples transferred to PVDF membranes was unaffected.CONCLUSION:Prestaining concen-trate gels with RBY or ERY can enhance the efficiency of gel-based electrophoresis and immunoblotting analysis.

Mulberry (Morus alba L.) leaf is a well-established traditional Chinese botanical and culinary resource. It has found widespread application in the management of diabetes. The bioactive constituents of mulberry leaf, specifically mulberry leaf flavonoids (MLFs), exhibit pronounced potential in the amelioration of type 2 diabetes (T2D). This potential is attributed to their ability to safeguard pancreatic β cells, enhance insulin resistance, and inhibit α-glucosidase activity. Our antecedent research findings underscore the substantial therapeutic efficacy of MLFs in treating T2D. However, the precise mechanistic underpinnings of MLF's anti-T2D effects remain the subject of inquiry. Activation of brown/beige adipocytes is a novel and promising strategy for T2D treatment. In the present study, our primary objective was to elucidate the impact of MLFs on adipose tissue browning in db/db mice and 3T3-L1 cells and elucidate its underlying mechanism. The results manifested that MLFs reduced body weight and food intake, alleviated hepatic steatosis, improved insulin sensitivity, and increased lipolysis and thermogenesis in db/db mice. Moreover, MLFs activated brown adipose tissue (BAT) and induced the browning of inguinal white adipose tissue (IWAT) and 3T3-L1 adipocytes by increasing the expressions of brown adipocyte marker genes and proteins such as uncoupling protein 1 (UCP1) and beige adipocyte marker genes such as transmembrane protein 26 (Tmem26), thereby promoting mitochondrial biogenesis. Mechanistically, MLFs facilitated the activation of BAT and the induction of WAT browning to ameliorate T2D primarily through the activation of AMP-activated protein kinase (AMPK)/sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) signaling pathway. These findings highlight the unique capacity of MLF to counteract T2D by enhancing BAT activation and inducing browning of IWAT, thereby ameliorating glucose and lipid metabolism disorders. As such, MLFs emerge as a prospective and innovative browning agent for the treatment of T2D.
Mice ; Animals ; Adipose Tissue, Brown ; Sirtuin 1/pharmacology* ; Diabetes Mellitus, Type 2/metabolism* ; AMP-Activated Protein Kinases/metabolism* ; Morus/metabolism* ; Flavonoids/metabolism* ; Prospective Studies ; Signal Transduction ; Adipose Tissue, White ; Plant Leaves ; Uncoupling Protein 1/metabolism* ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism*

【Objective】 To evaluate the efficacy and safety of human coagulation factor Ⅷ developed by Shenzhen Weiguang Biological products Co, Ltd in the treatment of patients with hemophilia A. 【Methods】 A prospective, multi-center, open, single-group clinical study was conducted. A total of 65 subjects with hemophilia A were enrolled, and human coagulation factor Ⅷ(FⅧ) was injected according to the patients’ bleeding severity. The improvement score of bleeding symptoms and signs after the first infusion of the first bleeding event and the transfusion efficiency of FⅧ activity at 10 min and 1 hour after infusion were taken as the main efficacy indexes. The improvement scores of bleeding symptoms and signs after the first infusion and the increase of FⅧ activity at 10 min and 1 hour after infusion were the secondary efficacy indexes. 【Results】 The 65 subjects were enrolled in safety analysis set (SS) and full analysis set (FAS), and 58 of them were enrolled in protocol analysis set (PPS). Ten minutes and one hour after the first infusion, the level of factor Ⅷ activity in the subjects increased significantly, and the FⅧ activity increased by 100% or more in more than 79% of the subjects. The average infusion efficiency of FⅧ activity in all subjects was more than 100%. In 70% of the subjects, the pain was relieved rapidly and /or the bleeding symptoms were significantly improved 8 hours after each bleeding infusion, and the improvement rate of bleeding symptoms and signs reached 100% 72 hours after infusion. 【Conclusion】 After infusion of human coagulation factor Ⅷ, the activity level of factor Ⅷ in patients with hemophilia A significantly increased. The infusion efficiency can reach a optimal level, and the bleeding symptoms can be significantly improved.

【Objective】 To evaluate the clinical efficacy and safety of one kind of human prothrombin complex concentrate in treatment of patients with hemophilia B. 【Methods】 The clinical data of 36 patients with hemophilia B treated with human prothrombin complex concentrate produced by Shenzhen Weiguang Biological Products Co. Ltd. from May 2018 to April 2019 were retrospectively analyzed, and its clinical efficacy and safety were analyzed. 【Results】 A total of 35 subjects entered the full analysis set (FAS)and safety set (SS), 33 subjects entered the per protocol Set (PPS). Thirty minutes after the first infusion of FAS subjects, the activity of coagulation factor Ⅸ increased from (3.93±0.975) IU/dL to (25.61±9.337) IU/dL, and the infusion efficiency was (96.43±22.007)%. The increased value of coagulation factor Ⅱ activity was (73.25±14.874) IU/dL. The activity of coagulation factor Ⅶ was (42.79±16.847) IU/dL. The increased value of coagulation factor Ⅹ activity was (65.29±17.042) IU/dL. The increased value of coagulation factor Ⅸ activity was (21.68±9.434%) IU/dL. Twenty-four hours after the first infusion of FAS subjects, the improvement of bleeding symptoms and signs was excellent in 21 cases (60%), improved in 14 cases (40.0%), and the effective rate was 100%. The incidence of adverse reactions was 2.9%(1/35), and there was no antibody to human coagulation factor Ⅸ and new virus infection. 【Conclusion】 Infusion of human prothrombin complex concentrate produced by Shenzhen Weiguang Biological Products Co. Ltd. in the treatment of hemophilia B has significant clinical efficacy and good safety.

Objective:To investigate the clinical effect of Dynamic neutralization system applied to the treatment of lumbar degenerative diseases with fatty infiltration of multifidus muscle.Methods:From Jan 2015 to Dec 2017, a total of 53 patients of lumbar degenerative diseases with multifidus fatty infiltration treated by Dynesys in our hospital were analyzed, included 21 males and 32 females, aged 66.2±7.4 (range 48-81) years. There were lumbar spinal stenosis in 37 casesand lumbar disc herniationin 16 cases; the index level included L 2-S 1 in 3 cases, L 3-S 1 in 13 cases, L 2-L 5 in 5 cases, L 4-S 1 in 17 cases, and L 3-L 5 in 15 cases. The pedicle screws were inserted at the point of intersection of the outer edge of superior articular process and the midline of transverse process. After discectomy of herniated disc and hyperplastic ligamentum flavum, the distance between the upper and lower pedicle screws was measured and then the spacer of the corresponding length was cut out. Finally, the spacer was placed and fixed between the upper and lower pedicle screws by the elastic rope. The degree of multifidus fat infiltration, lumbar lordosis (LL), pelvic incidence (PI), pelvic tilt (PT), sacral slop (SS), range of motion (ROM), intervertebral height (IH), Japanese Orthopaedic Association (JOA) score, Oswestry disability index (ODI), the MOS 36-item short-form health survey (SF-36) and visual analog scale (VAS) were evaluated postoperatively. Results:The operation was performed successfully in all the patients. The operation duration was 173.5±64.7 (range 125-240) min. Intraoperative blood loss was 469.5±118.2 (range 380-620) ml. The patients were followed up for 47.9±6.7 (range 38-62) months averagely. At the last follow-up, the degree of fatty infiltration of the multifidus muscle showed no further progress by MR scan. There was no significant difference in ROM and IH at different time points preoperativelyand postoperatively. The LL recovered from 37.6°±8.8° to 43.2°±9.1°, the PT decreased from 24.7°±9.3° to 20.5°±5.1°, and the SS increased from 22.1°±7.7°to 26.3°±8.0°. The JOA score increased from preoperative 6.4±1.2 to 20.6±2.8, ODI decreased from preoperative 50.6%±11.3% to 13.0%±3.4%, SF-36 increased from preoperative 81.5±3.6 to 95.5±4.2, and the VAS decreased from preoperative 4.2±1.0 to 1.1±0.6. One patient experienced loosening and displacement on the left side pedicle screw of the L2 vertebral body 3.5 years after operation, and herclinical symptom improved significantly after conservative treatment.Conclusion:Dynesysis is safe and effective for the treatment of lumbar degenerative diseases with fatty infiltration of multifidus muscle, and it can restore the complete structure and function of tension band at lower back and prevent the progress of multifidus muscle fat infiltration combined with postoperative rehabilitation training.

【Objective】 To establish and evaluate a nephelometric assay for the determination of immunoglobulin A (IgA) residues in human intravenous immunoglobulin(IVIG). 【Methods】 BN ProSpec^© automatic protein analyzer and its supporting immunoglobulin A determination kit (nephelometry) produced by German Siemens and the national standard of human IgA were used to establish the nephelometric assay to determine IgA residue in test products and verify the methodology. The test products include IVIG (pH4) prepared by low-temperature ethanol protein separation process and a novel IVIG prepared by chromatography. 【Results】 The average deviation of three calibration curves for IgA residues determination by the nephelometric assay were 1.08%, 0.95% and 1.54%,, and the three deviations of the quality control were 4.00%, -2.30% and -0.20%, respectively, which indicated good calibration and quality control. In the specificity test, the average recovery rates of IgA for reference substance 1 containing 100g/L maltose and reference substance 2 containing 20g/L glycine were 102.7% and 105.8%, respectively. The relative standard deviation (RSD) values of the repeatability tests of the two test products were 3.9% and 1.9%, and the RSD values of the intermediate precision test were 3.6% and 2.3%, respectively.The difference values at each time point in the durability test of test products′ storage time were all less than 10%, and the RSD values of the two test products in the durability test of kits of different batches were 2.8% and 2.2%, respectively. In the accuracy test, the average recovery rates of IVIG (pH4) added to the standard were 94.2%, 101.7% and 96.2%, respectively, and the average recovery rates of the novel IVIG added to the standard were 102.8%, 106.3% and 99.7%, respectively. The average recovery rate of the limit quantification test was 101.0%, and the RSD was 4.0%. 【Conclusion】 Nephelometric assay has the advantages of strong specificity, high precision and accuracy, good repeatability, simple and rapid operation, and automation, and can be used for the determination of IgA residue in IVIG (pH4) and novel IVIG products.

Objective:To explore the mechanism of HDAC6 mediated aggresome-autophagy-lysosome pathway in paraquat-induced autophagy in dopaminergic neurons.Methods:Human neuroblastoma cell (SH-SY5Y cell) was used as model of dopaminergic neurons in vitro. The cells were treated with terminal concentrations of 0, 25, 50, 100, 200 and 400μmol/L PQ for 24 hours, and the cells were induced by 100 μmol/L PQ for different time (0, 12, 24, 36, 48, 60 and 72 h) . Cell viability was detected by CCK-8 assay. The expression levels of HDAC6, α-syn, Dynein IC1/2, LC3, Beclin1, p62 and Lamp-1 were detected by Western blot. Immunofluorescence double-labeling method was used to observe the expression and localization of HDAC6, α-syn, Dynein IC1/2, LC3, Lamp-1 and γ-tubulin in cells.Results:CCK-8 assay showed PQ induced cell survival rate decrease in a time and dose dependent manner ( R=-0.950、-0.960, P<0.05) .Western blot showed that compared with control group, the protein levels of HDAC6, α-syn, p62 in PQ-exposed group were significantly increased ( P<0.05) , but there was a significant decrease in expression level of the ratio of autophagy-related protein LC3 Ⅱ/LC3 Ⅰ, Beclin1, Dynein IC1/2, Lamp-1in PQ-exposed group ( P<0.05) . The results of immunofluorescence double-labeling showed that compared with the control group, the fluorescence signals of HDAC6 and α-syn in the PQ-exposed group increased, and the protein expression level increased, while the fluorescence signals of Dynein IC1/2, LC3, and Lamp-1 decreased. The protein expression level is reduced. HDAC6 gradually accumulates from the diffuse shape to the nucleus; Under normal circumstances, α-syn, Dynein IC1/2, γ-tubulin, LC3, and Lamp-1 are mainly distributed in the cytoplasm. After PQ is infected, they gather in the nucleus and co-localize with HDAC6 in the area around the nucleus. Conclusion:PQ may induce abnormal aggregation of α-syn by inducing HDAC6-mediated aggresome-autophagy-lysosomal pathway disorder.

Clostridioides difficile is a key pathogen of antibiotic related diarrhea and hospital associated infection, causing several outbreaks in Europe and North Americans and resulting in severe disease burden. However, the standardized diagnostic principle and detection specifications in C. difficile infection (CDI) survey are limited in China, and the infection rate and disease burden of CDI in China are unclear. Therefore, National Institute for Communicable Disease Control and Prevention,National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, together with another 11 institutions, draft the group standard entitled "Diagnosis of Clostridium difficile infection (T/CPMA 008-2020)" of Chinese Preventive Medicine Association. Based on the principle of "legality, scientificity, advancement, and feasibility", this standard clarifies risk factors, diagnosis principles, diagnoses and differential diagnoses in order to improve the accuracy of CDI diagnosis in clinical practice, guide the surveillance for CDI, and understand the infection rate and disease burden of CDI in China .

Objective:To explore the mechanism of HDAC6 mediated aggresome-autophagy-lysosome pathway in paraquat-induced autophagy in dopaminergic neurons.Methods:Human neuroblastoma cell (SH-SY5Y cell) was used as model of dopaminergic neurons in vitro. The cells were treated with terminal concentrations of 0, 25, 50, 100, 200 and 400μmol/L PQ for 24 hours, and the cells were induced by 100 μmol/L PQ for different time (0, 12, 24, 36, 48, 60 and 72 h) . Cell viability was detected by CCK-8 assay. The expression levels of HDAC6, α-syn, Dynein IC1/2, LC3, Beclin1, p62 and Lamp-1 were detected by Western blot. Immunofluorescence double-labeling method was used to observe the expression and localization of HDAC6, α-syn, Dynein IC1/2, LC3, Lamp-1 and γ-tubulin in cells.Results:CCK-8 assay showed PQ induced cell survival rate decrease in a time and dose dependent manner ( R=-0.950、-0.960, P<0.05) .Western blot showed that compared with control group, the protein levels of HDAC6, α-syn, p62 in PQ-exposed group were significantly increased ( P<0.05) , but there was a significant decrease in expression level of the ratio of autophagy-related protein LC3 Ⅱ/LC3 Ⅰ, Beclin1, Dynein IC1/2, Lamp-1in PQ-exposed group ( P<0.05) . The results of immunofluorescence double-labeling showed that compared with the control group, the fluorescence signals of HDAC6 and α-syn in the PQ-exposed group increased, and the protein expression level increased, while the fluorescence signals of Dynein IC1/2, LC3, and Lamp-1 decreased. The protein expression level is reduced. HDAC6 gradually accumulates from the diffuse shape to the nucleus; Under normal circumstances, α-syn, Dynein IC1/2, γ-tubulin, LC3, and Lamp-1 are mainly distributed in the cytoplasm. After PQ is infected, they gather in the nucleus and co-localize with HDAC6 in the area around the nucleus. Conclusion:PQ may induce abnormal aggregation of α-syn by inducing HDAC6-mediated aggresome-autophagy-lysosomal pathway disorder.