AIM To establish an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous content determination of gallic acid,matrine,danshensu,hydroxysafflor yellow A,caffeic acid,paeoniflorin,ferulic acid and cynaroside in Xuebijing Injection (Carthami Flos,Paeoniae Radix Rubra,Chuanxiong Rhizoma,etc.).METHODS The analysis of 5％ acetonitrile extract of this drug was performed on a 40 ℃ thermostatic ACQUITY UPLC(C) BEH C18 column (2.1 mm × 50 mm,1.8 μm),with the mobile phase comprising of acetonitrile-0.1％ formic acid flowing at 0.2 mL/min in a gradient elution manner,after which cluster analysis was applied to the determined contents.RESULTS Eight constituents showed good linear relationships within their own ranges (r ＞ 0.996 0),whose average recoveries were 95％-104％ with the RSDs of less than 3％.Generally the contents of various constituents in ten batches of samples were found to be stable except the certain differences contributed by gallic acid and paeoniflorin.CONCLUSION We should pay attention to the quality of different batches of Xuebijing Injection due to their uneven performance.

Objective To find the different plasma-associated proteins of rheumatoid arthritis (RA) by using two-dimensional gel electrophoresis for understanding the pathogenesis of RA. Methods The total protein from either RA patients or normal ones was prepared by means of immobilized pH gradient based on two-dimensional gel electrophoresis. After silver staining, gel-image analysis was performed by using PDQuest. The differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). Results 2-DE patterns of plasma from controls and RA patients were presented. The results showed that average number of protein spots was 592 and 563 respectively, and the corresponding average matching rate was 89% and 87% respectively. Gel-image analysis revealed that there were 24 differential protein spots. A total of 15 differential protein spots were successfully identified by MALDI-TOF-MS, of which 6 proteins were up-regulated as compared with control. Conclusion The differentially expressed proteins can be observed in plasma from RA and controls, which can be used to elucidate the pathogenesis of RA for further study.

ObjectiveTo understand the possible role of some proteins expressed by human synovial fibroblasts(SFs)in the pathogenesis of rheumatoid arthritis.MethodsThe expression difference of synovial fibroblast proteins between rheumatoid arthritis(RA)patients and healthy controls was analyzed by 2-DE.The differential expression spots were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry(MALDI-TOF-MS),followed by bioinformatics analysis,some of which were validated by Western blot.ResultsUsing 12% SDS-PAGE following pH 4-7 IPG strips in IEF,averagely 837 and 852 protein spots were detected in RA patients and normal subjects,respectively.Gel image analysis revealed that there were 49 differential protein spots.By peptide mass fingerprinting strategy,we identified 40 protein spots derived from gels of SFs in RA patients among differential spots and 23 valid proteins were obtained.Western blot analysis showed that expressions of Enolase ?,Annexin I,Cathepsin D,SOD2,Peroxiredoxin 2 were significantly higher in SFs from RA patients than those from normal subjects,which was consistent with proteome analysis.ConclusionThe differential proteins might be involved in inflammation of synovitis in RA.

Objective To screen the proteins interacting with FOXP3 in yeast two-hybrid system. Methods The "bait plasmid" pGBKT7 (named as pGBKT7-FOXP3) was constructed successfully. Using FOXP3 as bait, a human liver cDNA library was screened and the proteins interacting with FOXP3 were searched. The false positive clones were discarded by one to one yeast two-hybrid system, and the positive clones were sequenced and analyzed by bioinformatic methods. Results The bait plasmid pGBKT7-FOXP3 was constructed successfully and there was no self-activation or toxicity in AH109. Three proteins had been found in our system to be able to interact with FOXP3. They were tumor protein D52, splicing factor 3b subunit 1 and one hypothetical protein. Conclusion FOXP3 interacts with tumor protein D52, splicing factor 3b subunit 1 and one hypothetical protein, all of which may interfere in cell metabolism and function of T cell.

Objective: To explore the relationship of discrimination perception and social anxiety of the left-behind children, focusing on the moderating effect of trait gratitude. Methods: Totally 588 left-behind children were investigated with discrimination perception questionnaire, gratitude questionnaire and Liebowitz social anxiety questionnaire. Data were analyzed by independent t test, correlation analysis and regression analysis. Results: There was a significant difference in group discrimination between the only child (2.11±0.98) and the non-only child (2.28±0.93) (t=-2.11, P<0.05). Correlation analysis showed that trait gratitude (4.93±0.89) was negatively correlated with individual discrimination (2.53±1.03) and group discrimination (2.22±0.95) (r=-0.55, r=-0.56, both P< 0.05), furtherly negatively correlated with fear of negation (0.85±0.47) and social avoidance (0.81±0.53) dimensions of social anxiety (r=-0.47, r=-0.48, both P<0.05). Additionally, discrimination perception was positively correlated with social anxiety (r=0.61, P<0.05). Regression analysis showed that discrimination perception could significantly predict social anxiety (β=0.44, P<0.01), and trait gratitude could significantly moderate this effect (β=-0.75, P<0.01). Conclusion Discrimination perception can positively predict social anxiety, and trait gratitude can moderate this effect.

To explore the role of dentritic epidermal T lymphocytes ( DETCs) in immune rejection of skin allograft in mice and its related mechanism. Methods (1) Full-thickness skin was harvested from back of one male wild type (WT) C57BL/6 mouse. Epithelial cells were isolated for detection of the expression of DETCs and their phenotype with flow cytometer. Another male WT C57BL/6 mouse was used to harvest full-thickness skin from the back. Epidermis was isolated for observation of the morphological characteristics of DETCs with immunofluorescence technology. (2) Four male green fluorescence protein (GFP)-marked C57BL/6 mice, 7 female WT C57BL/6 mice (group WT), and 7 female ybT lymphocytes 8 gene knock-out (GK) C57BL/6 mice (group GK) were used. Full-thickness skin in the size of 1.4 cm x 1.4 cm on the back of mice in groups WT and GK were excised, and the wounds were transplanted with full-thickness skin in the size of 1.2 cm x 1.2 cm obtained from male GFP-marked C57BL/6 mice. The survival time of skin grafts was affirmed with small animal in vivo imager and naked eyes and recorded. (3) Two male WT C57BL/6 mice were used to isolate epithelial cells. Cells were inoculated into 48-well plate and divided into activation group (A) and control group (C) according to the random number table, with 4 wells in each group. Cells in group A were treated with 10 pL concanavalin A in the concentration of 2 microg/mL for 24 hours, while those in group C with PBS in the same volume as that in group A. The expression of interferon y in DETCs was detected with flow cytometer. (4) Four male GFP-marked C57BL/6 mice were used as donors. Fourteen female WT C57BL/6 mice were used as receptors and divided into interferon gamma neutralizing group (IN) and control group (C) according to the random number table, with 7 mice in each group. The skin transplantation model of C57BL/6 male to C57BL/6 female was established as in part (2). Before surgery and 72 hours after, mice in group IN were intraperitoneally injected with 200 pL interferon y neutralizing antibody in the concentration of 1 mg/mL, and those in group C with normal saline in the same volume as that in group IN. The survival time of skin grafts was observed and recorded using the methods in part (2), and the result of group IN was compared with that of group GK in part (2). The survival curve of skin grafts was processed with Log-rank ( Mantel-Cox) test. Results (1) The positive expression rate of DETCs in epithelial cells of skin in mouse was 7.27%, and they were all CD3 cells. DETCs were found to be scattered in the epidermis of skin in mouse with dendritic morphology. (2) The survival time of skin grafts of mice in group GK was 22-35 d, obviously longer than that in group WT (12-16 d, y2 = 14. 10 , P < 0.001). (3) Expression of interferon gamma was detected in 22. 70% DETCs in group A, which was obviously higher than that in group C (0.51%). (4) The survival time of skin grafts of mice in group IN was 19-24 d, which was obviously longer than that in group C (12-16 d, chi 2 = 13.60, P < 0.001) but close to that in group GK as in part (2) (chi2 = 0.06, P = 0.810). Conclusions DETCs are involved in promotion of immune rejection of skin allograft probably by secretinf interferon gamma.
Allografts ; Animals ; Epidermis ; Female ; Graft Survival ; immunology ; physiology ; Interferon-gamma ; immunology ; metabolism ; Lymphocytes ; Male ; Mice ; Mice, Inbred C57BL ; Skin ; Skin Transplantation ; T-Lymphocytes ; immunology

Objective To explore the relationship of discrimination perception and social anxiety of the left-behind children,focusing on the moderating effect of trait gratitude. Methods Totally 588 left-be-hind children were investigated with discrimination perception questionnaire, gratitude questionnaire and Liebowitz social anxiety questionnaire. Data were analyzed by independent t test,correlation analysis and re-gression analysis. Results There was a significant difference in group discrimination between the only child (2. 11±0. 98) and the non-only child (2. 28±0. 93) (t=-2. 11,P<0. 05). Correlation analysis showed that trait gratitude (4. 93± 0. 89) was negatively correlated with individual discrimination ( 2. 53 ± 1. 03) and group discrimination (2. 22±0. 95) (r=-0. 55,r=-0. 56,both P< 0. 05),furtherly negatively correlated with fear of negation (0. 85±0. 47) and social avoidance (0. 81±0. 53) dimensions of social anxiety ( r=-0. 47,r=-0. 48,both P<0. 05). Additionally,discrimination perception was positively correlated with so-cial anxiety (r=0. 61,P<0. 05). Regression analysis showed that discrimination perception could significant-ly predict social anxiety (β=0. 44,P<0. 01),and trait gratitude could significantly moderate this effect (β=-0. 75,P<0. 01). Conclusion Discrimination perception can positively predict social anxiety,and trait gratitude can moderate this effect.

Objective To construct a bait vector containing human Foxp3 gene in yeast two-hybrid system in order to screen the cDNA library of T lymphocyte. Methods RT-PCR was used to amplify the Foxp3 gene fragment from the peripheral blood mononuclear cells (PBMC) with the primers designed in accordance with the sequence in GenBank. The product was inserted into pMD18-T vector. After verified with restriction endonuclease digestion of EcoRⅠ and SalⅠ, the vector was inserted into the “bait plasmid” pGBKT7 (named as pGBKT7-Foxp3). After confirmation with restricted endonuclease digestion and sequence analysis, the plasmid was transformed into the yeast cell AH109, and its toxicity and transcriptional activation was tested by both the phenotype assay and the color assay. Results The amplified product of 1 203 bp was inserted into PMD18-T vector and proven correctly by double restriction enzyme digestion. Sequence analysis revealed that the fragment was correctly inserted into pGBKT7 with a right reading frame and its expression in yeast was verified. Conclusion The bait plasmid pGBKT7-Foxp3 constructed expresses correctly, and can not activate the transcription of reporter gene alone in yeast two-hybrid system

Objective To explore the interaction between HT036(hypothetical protein HT036)and P311 by co-immunoprecipitation.Methods HA-tagged fusion protein(HA-HT036)expression vector was constructed,identified and transfected into human embryo kidney 293(HEK293)cells alone or with Myc-tagged fusion protein(Myc-P311)expression vector pCMV-Myc-p311.The interaction between P311 and HT036 was detected by co-immunoprecipitation.Results Double restriction enzyme digestion showed that pCMV-HA-HT036 was constructed correctly.When Myc-P311 was immunoprecipitated by anti-Myc antibody,HA-HT036 was identified by Western blotting with anti-HA antibody from immunoprecipitated complex.Conclusion The recombinant vector pCMV-HA-HT036 was constructed successfully.The interaction between HT036 and P311 could be identified by co-immunoprecipitation after co-expression of pCMV-HA-HT036 and pCMV-Myc-p311.The result provides an important basis for further study of the intracellular signal transduction of P311.

2019 novel coronavirus (2019-nCoV) is one of the beta coronaviruses and was identified as the pathogen of the severe "coronavirus disease 2019 (COVID-19)" in 2019. China has formally included the 2019-nCoV in the statutory notification and control system for infectious diseases according to the Law of the People's Republic of China on the Prevention and Treatment of Infectious Diseases. Currently, the national defending actions on the 2019-nCoV in China is in a critical period. Burn Department is also confronted with risk of infection by the 2019-nCoV. According to the guidelines on the diagnosis and treatment of COVID-19 (6^th trial edition), the latest relative literature at home and abroad, the features of the COVID-19, recommendations for the COVID-19 prevention and control issued by the National Health Commission of China, and management experience of diagnosis and treatment in the related disciplines, we put forward recommendations for the medical practices of burn treatment during the outbreak of the COVID-19 in outpatient and emergency treatment, inpatient treatment, operation and ward management, etc. We hope these recommendations could benefit the professionals of the same occupation as us and related hospital managers, improve the treatment of burn during the outbreak of the COVID-19, and avoid or reduce the risk of infection of medical staff .