Objective To study the effect of Salidroside(Sal)on platelet activation and aggregation and the interaction between human platelets and MDA-MB-468 cells of breast cancer.Methods Human platelets were collected,platelet sus-pension was prepared,and platelets were treated with different concentrations of Sal.The effects of Sal on platelet activation and aggregation were detected by thromboelastogram(TEG)and flow cytometry.Breast cancer MDA-MB-468 cells were cul-tured in vitro,and human platelets were treated with different concentrations of Sal,and then activated by thrombin.The effects of Sal on the interaction between platelets and MDA-MB-468 cells were analyzed by adhesion test and scratch test.Re-sults TEG detection:The ADP inhibition rate in the blank control group was(10.97±12.69)%,and the ADP inhibition rate in all Sal intervention groups was higher than that in the blank control group(P＜0.05).The AA inhibition rate was(8.11±7.84)%in the control group and(25.96±15.18)%in the 5 μmol/L Sal intervention group,and the difference was statistically significant(P＜0.05).Flow cytometry:The expression of CD62P on platelet surface in 40 and 60 μmol/L Sal groups was(56.5±0.17)%and(65.50±0.36)%,respectively,and the difference was statistically significant compared with the positive control group(76.53±0.49)%(P＜0.05).The percentages of PAC-1 expression on platelet surface in 40 and 60 μmol/L Sal groups were(62.20±0.10)%and(58.47±0.15)%,and the difference was statistically significant com-pared with the positive control group(72.10±0.20)%(P＜0.05).Adhesion experiment:Platelets can have adhesion with MDA-MB-468 cells,and activated platelets have stronger adhesion ability.The adhesion rate in the Sal group was signifi-cantly lower than that in the positive control group,and was negatively correlated with the concentration of Sal.Scratch test:The cell mobility at 24 h in the positive control group was(12.71±0.70)%,and the cell mobility in each Sal treatment group was(4.51±0.44)%,(3.85±0.11)%,(5.37±0.36)%,(4.15±0.13)%and(3.55±0.38)%,respectively,showing significant decrease compared with the positive control group(P＜0.05).After 48 h of Sal treatment,the cell mobility of 10,20,40 and 60μmol/L Sal groups decreased,and there was a statistical difference compared with the positive control group(P＜0.05).Conclusion Sal can inhibit the adhesion between activated platelets mediated by thrombin and MDA-MB-468 cells and the migration of MDA-MB-468 cells.

Objective:To evaluate effect of diammonium glycyrrhizinate(DG)on lipopolysaccharide(LPS)-induced TLR4/NF-κB inflammatory signaling pathway in BV2 microglial cells and to explore its potential regulatory mechanisms on ameliorating neu-roinflammation.Methods:BV2 microglia injury model was induced by LPS and treated with 20 and 60 μmol/L DG.MTS was used to determine vitality of cells.NO level secreted by cells was detected by Griess.Inflammatory factors TNF-α,IL-6,and IL-1β levels in cell supernatant were measured by ELISA.TNF-α,IL-6,IL-1β,TLR4 and MyD88 mRNA levels were detected by RT-qPCR.Western blot was used to determine expressions of TLR4,MyD88,NF-κB,p-NF-κB,IκB-α and p-IκB-α proteins of cells.Results:Compared with control group,LPS-treated BV2 microglia had significantly lower viability(P<0.05),significantly higher NO secretion(P<0.05),significantly up-regulation levels of inflammatory factors TNF-α,IL-6 and IL-1β(P<0.05),and significantly higher mRNA levels of TNF-α,IL-6,IL-1β,TLR4 and MyD88(P<0.05),TLR4,MyD88,NF-κB,p-NF-κB,IκB-α,and p-IκB-α protein expressions were significantly increased(P<0.05).Compared with LPS group,BV2 microglia intervened with different concentrations of DG had significantly higher viability(P<0.05),significantly lower NO secretion(P<0.05),significantly lower TNF-α,IL-6 and IL-1β inflammatory factors levels(P<0.05),significantly lower mRNA levels of TNF-α,IL-6,IL-1β,TLR4 and MyD88(P<0.05),and TLR4,MyD88,p-NF-κB and p-IκB-α protein expressions were significantly reduced(P<0.05).Conclusion:DG can inhibit LPS-induced neuroinflammatory responses in microglia,whose underlying mechanism is suppression of TLR4/NF-κB signaling pathway.

Objective To retrospectively analyze the diagnostic value of thrombin-antithrombin complex(TAT),thrombomodulin(TM),and tissue plasminogen activator-inhibitor complex(t-PAIC)in severe cases of corona virus disease 2019(COVID-19).Methods A cohort of 79 patients clinically diagnosed with COVID-19 was retrospectively assembled and categorized into two groups based on disease severity:non-severe(n=51)and severe(n=28).In this study the differences of coagulation function and inflamma-tory marker levels between the two groups were compared.The correlations of TAT,TM and t-PAIC with other biomarkers were investi-gated.The diagnostic values of all the markers for severe COVID-19 were assessed.Results The patients of severe COVID-19 exhibi-ted significantly higher levels of TAT,TM,and t-PAIC compared to those of non-severe group(P＜0.001).The levels of TAT,TM and t-PAIC showed notable positive correlation with other biomarkers.TAT demonstrated the strongest positive correlation with the level of D-dimer(r=0.786,P＜0.001).Binary logistic regression analysis identified TAT(OR=1.346,P＜0.05)and t-PAIC(OR=1.128,P＜0.05)were independent risk factors in term of severe COVID-19.The combined ROC curve for TAT,TM and t-PAIC revealed high diagnostic efficacy in severe cases with the area under the curve(AUCROC)were 0.918,and the sensitivity and specificity were of 75％and 94.1％,respectively.Conclusion The results of combined measurement of TAT,TM and t-PAIC effectively demonstrates its diagnostic value in identifying severity and stratification of COVID-19 cases and may have important clinical significance for assessment of the severity and prediction of the prognosis.

【Objective】 To investigate the feasibility of allogeneic platelet-rich plasma (PRP) for the treatment of herpes zoster wounds secondary to systemic lupus erythematosus (SLE), especially large ulcer wounds. 【Methods】 The treatment process of a patient with massive herpes zoster wounds in perineum and hip accompanied by extensive soft tissue necrosis secondary to SLE was retrospectively analyzed. The clinical efficacy of allogeneic PRP was explored combined with treatment key points and literature review. 【Results】 The patient′s wound bed was prepared until the wound was fresh, then treated externally with allogeneic PRP 3 times a week. The wound was healed completely after 42 days. 【Conclusion】 In the case of autologous PRP unavailable or unsuitable, allogeneic PRP is a safe alternative, which can effectively promote tissue regeneration, and this patient achieved curative effect in a short period of time.

Neuroendocrine tumors of the gallbladder(GB-NET) are rare, and it lacks early clinical manifestations and has no specific tumor markers, it is difficult to distinguish GB-NET from gallbladder adenocarcinoma. The diagnosis of GB-NET is based on histopathology of the tumor and the assessment of proliferation fraction, which makes it difficult to achieve early diagnosis. GB-NET has a high degree of malignancy, 32.39% of patients have liver metastases at diagnosis, and 51.60% of patients have lymph node metastases, the median survival time is 9 to 10 months.There are currently no specific guidelines or consensus for the treatment of GB-NET. The treatment strategies are choosen mainly by the principles of gallbladder adenocarcinoma. We reviews the clinical and basic researches of GB-NET and case reports from China and across the world, as well as the data from SEER database, and we discuss the research progress on the classification, clinicopathological features, diagnosis, treatment advances and the prognosis.

【Objective】 To investigate the effect of leukocyte filter in removal of tumor cells in blood and the viability of using leukocyte filter in intraoperative cell salvage in tumor patients. 【Methods】 The leucocyte-depleted suspended RBCs prepared from 200 mL human whole blood were spiked with HCT116 cells cultured in vitro to simulate the autologous blood recovered from tumor patients during operation. Conventional filter (group A, n=6), filter with small pore size (group B, n=6) and filter with thicker membrane and small pore size (group C, n=6)) were used. The CD45^-EpCAM⁺ tumor cells were detected by anti-CD45-PE and anti-CD326 (EpCAM)-FITC flow cytometry, and the absolute count of tumor cells were calculated using Flow Count fluorescence microspheres. The activity of tumor cells was detected by cell culture and trypan blue staining. Furthermore, blood biochemical indexes (LDH, K⁺ ) and red cell recovery rate were detected. 【Results】 Tumor cell counts before and after the application of leukocyte filter A, B, C were(1.52±0.48)×10⁷/mL vs(2.73±1.74)×10⁴/mL, (1.48±0.55)×10⁷/ml vs(2.96±1.85)×10⁴/mL and (1.44±0.46)×10^7/mL vs(3.08±2.33)×10⁴/mL, respectively (P >0.05), which was significantly reduced after filtration (P < 0.01). No viable cells were found in the filtered blood cultured for 7 days. There were no significant differences in K⁺ and LDH value before and after filtration among the three groups(P >0.05). The blood recovery rates of group A, B, C were 85.22%, 84.97% and 82.86%, respectively. 【Conclusion】 The conventional filter can significantly reduce the number of circulating tumor cells, and it is feasible for intraoperative cell salvage in tumor patients.

The capacity for thermal tolerance is critical for industrial enzyme. In the past decade, great efforts have been made to endow wild-type enzymes with higher catalytic activity or thermostability using gene engineering and protein engineering strategies. In this study, a recently developed SpyTag/SpyCatcher system, mediated by isopeptide bond-ligation, was used to modify a rumen microbiota-derived xylanase XYN11-6 as cyclized and stable enzyme C-XYN11-6. After incubation at 60, 70 or 80 ℃ for 10 min, the residual activities of C-XYN11-6 were 81.53%, 73.98% or 64.41%, which were 1.48, 2.92 or 3.98-fold of linear enzyme L-XYN11-6, respectively. After exposure to 60-90°C for 10 min, the C-XYN11-6 remained as soluble in suspension, while L-XYN11-6 showed severely aggregation. Intrinsic and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence analysis revealed that C-XYN11-6 was more capable of maintaining its conformation during heat challenge, compared with L-XYN11-6. Interestingly, molecular cyclization also conferred C-XYN11-6 with improved resilience to 0.1-50 mmol/L Ca²⁺ or 0.1 mmol/L Cu²⁺ treatment. In summary, we generated a thermal- and ion-stable cyclized enzyme using SpyTag/SpyCatcher system, which will be of particular interest in engineering of enzymes for industrial application.
Animals ; Cyclization ; Endo-1,4-beta Xylanases ; chemistry ; metabolism ; Enzyme Stability ; Industrial Microbiology ; methods ; Microbiota ; Protein Engineering ; Rumen ; enzymology ; microbiology ; Temperature

Objective To develop the automated preparation of 18F-Alfatide II using newly-designed 18F-minireactor and perform 18F-Alfatide D microPET/CT imaging in tumor.Methods The automated preparation of 18F-Alfatide H was developed by using 18F-microreactor and water phase Al18F-chelating method,and the radiochemical yield and quality analysis were measured.The nude mice bearing breast tumor ZR-75-1 and nasopharyngeal tumor CNE1 were established(n = 3 respectively).MicroPET/CT imaging was performed at 0.5,1.0 and 2.0 h after the injection of 18F-Alfatide II.The region of interest(ROI)was depicted and the tumor/muscle(T/M)ratio was calculated.Results 18F-Alfatide II was automatically prepared with the total synthesis time of 40 min,the radiochemical yield of(28±6)%(no decay corrected,n=11),and the radiochemical purity >97%.All quality analysis indexes accorded with the radiopharmaceutical requirements.18F-Alfatide II microPET/CT images of ZR-75-1 and CNE1 tumors were clear due to the high radioactivity uptake of tumor lesions(T/M ratio was greater than 4.0 at 1.0 h after injection).Conclusion Based on the 18F-minireactor,the,8F-Alfatide II can be prepared successfully with short synthesis time and high radiochemical yield,which can help the application studies in 18F-Alfatide II microPET/CT imaging.

Objective: To explore the method of the blood return of continuous blood purification therapy for patients with heart failure. Methods: A total of 69 patients with heart failure treated by continuous blood purification therapy in our ICU from January 2017 to January 2018 were randomly divided into experimental group (blood return with blood bag,35 patients) and the control group (blood return with machine,34 patients). The vital signs, clinical manifestations and hemodynamic indicators of each patient were collected during the blood return of the first CBP therapy, and the data were analyzed between the two groups. Results: After blood return of the first CBP therapy, the exacerbation of New York Heart Association (NYHA) heart function grade, SpO₂, CVP, HR of patients in the control group were 33.27% (11/31), 0.91 ± 0.06, (12.44 ± 1.43) cmH₂O (1cmH₂O=0.098 kPa), (118.17 ± 3.27) times per minute and those in the experimental group were 6.25% (2/32), 0.96±0.04, (8.98±1.36) cmH₂O, (90.45 ± 3.35)times per minute, respectively. The difference was statistically significant between the two groups (χ²=3.786, t=2.861, -7.565, 3.792, P< 0.05). Subgroup analysis showed that for patients within 24 hours of CBP treatment, the exacerbation of NYHA heart function grade, SpO₂, CVP, HR of the control group and the experimental group were respectively 76.92% (10/13), 0.86±0.01, (12.92±1.12) cmH₂O, (111.38±2.96) times per minute and 8.33% (1/12), 0.94±0.01, (8.11±0.74) cmH₂O, (90.34±1.32) times per minute, the difference was statistically significant (χ² =9.345, t=-14.101, 2.894, 7.648, P < 0.05). For patients treated with CBP therapy over 24 hours, the difference of the exacerbation of NYHA heart function grade, SpO₂, CVP, HR of the control group and the experimental group was not statistically significant (P>0.05). Conclusions In patients with heart failure treated with CBP, especially in those treated with CBP therapy within 24 hours, blood return with blood bag may avoid the recurrence of heart failure.

Isopeptide bond-mediated molecular superglue is the irreversible covalent bond spontaneously formed by the side chains of lysine (Lys) and asparagine/aspartic acid (Asn/Asp) residues. The peptide-peptide interaction is specific, stable, and can be achieved quickly without any particular physicochemical factor. In the light of recent progress by domestic and foreign researchers, here we summarize the origin, assembly system and mechanism of isopeptide bond reaction, as well as the molecular cyclization and protein topological structure mediated by it. The prospect for its application in synthetic vaccine, hydrogel and bacterial nanobiological reactor is further discussed.
Cyclization ; Lysine ; Peptides ; chemistry ; Proteins