Objective To investigate the repair and protective effect of extracorporeal membrane oxygenation (ECMO) on liver and bile duct after cardiac death in pig.Methods Eight pigs were purchased and cardiac arrest was induced by the administration of 1 g KCL intravenously,followed by 30 min cardiopulmonary resuscitation according to standard guideline.Cannulas were placed through inferior vena cava and abdominal aorta,and then connected to ECMO extracorporeal circulation pipes.ECMO was performed for 4 h.Circulation flow rate of hepatic artery and bile production were monitored and recorded.Lactate dehydrogenase (LDH),γ-glutamyl transferase (γ-GT) and direct bilirubin (DBIL) in bile were detected.Transaminase,tumor necrosis factor-α (TNF-α),interleukin-1β (IL-13),hyaluronic acid (HA),endothelin-1 (ET-1) and nitric oxide (NO) in serum were detected.Pathological change was observed by HE staining under optical microscope and cell apoptosis was detected by TUNEL.Results There was no bile production after cardiac death,which increased to 80％ of the baseline after 4h of ECMO.In addition,γ-GT,LDH and DBIL content in bile was (23.3 ± 11.8) IU/L,(15.9 ± 3.3) IU/L and (72.3 ± 21.4) mmol/ L,and IL-1,TNF-α and HA content in serum was (117.6 ± 39.0) ng/L,(120.4 ± 16.5) ng/L and (63.7 ± 4.4) ng/L,respectively,and no statistically significant differences were observed when compared with the baseline (all P ＞ 0.05).ET-1 content was (4.9 ± 1.3) ng/L and NO content was (135.3 ± 16.7)mmol/L in serum,which was statistically increased (both P ＜ 0.05).Pathological changes of liver and bile duct were significantly alleviated.Conclusion ECMO could exert protective effect on liver and bile duct after cardiac death.

Objective To investigate the optimal time of extracorporeal membrane oxygenation (ECMO) on repairing Maastricht Ⅱ pig liver with warm ischemia injury for 30 min.Method Thirty-six miniature pigs were randomized to ECMO 2-h group,ECMO 4-h group and ECMO 6-h group,12 pigs per group,6 donors and 6 recipients.Cardiac arrest was induced by administration of 1 g KCl intravenously,followed by cardiopulmonary resuscitation (CPR) for 30 min according to the standard guideline.Cannulas was placed through inferior vena cava and abdominal aorta,then connected to ECMO extracorporeal circulation pipes.Transaminase,circulation flow rate of portal vein and hepatic artery and arterial blood gas were monitored and recorded.The hepatic tissues were cut into sections for pathological observation by HE stain under a light microscope.Apoptosis was detected by TUNEL and apoptotic index was calculated.The liver was stored in cold UW for 2 h after the ECMO circulation,then orthotopic liver transplantation without veno-venous bypass was performed.The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured in the peripheral blood for 5 days after the operation.Result With the increase of the running time of ECMO,transaminase and lactate levels were decreased continuously.Circulation flow rate of portal vein and hepatic artery in ECMO 6-h group was significantly lower than that in the other two groups (P＜0.05).Pathological change in ECMO 4-h group was milder than the rest two groups.Apoptosis index in ECMO 2-h,4-h and 6-h groups was (40.20 ± 7.22)％,(18.60 ± 4.04)％ and (29.25 ± 5.98) ％,respectively.The 5-day suvival rate in ECMO 2-h,4-h and 6-h groups was 83％,100％ and 83％,respectively.The transaminase level in ECMO 4-h group at 5th day after the operation was lower than in ECMO 2-h group and 6-h group (P＜0.05).Conclusion The optimal time of ECMO on repairing Maastricht Ⅱ liver was 4 h.The effect of restoration is not ideal when circulation time is not enough.Liver function and liver cell viability decline beyond 4 h.

Objective To study the repair mechanism of extracorporeal membrane oxygenation on liver after cardiac death.Methods Twelve pigs were equally randomized to ECMO group and control group.Cardiac arrest was induced by administration of 1 g KCL intravenously,followed by 30 min cardiopulmonary resuscitation according to the standard guideline Cannulas were placed through inferior vena cava and abdominal aorta,then connected to ECMO extracorporeal circulation pipes in ECMO group for 4 h.The livers were stored in cold UW for 4 h in control group.ATP,superoxide dismutase (SOD),glutathionein (GSH),malondialdehyde (MDA),heat shock protein 70 (HSP70) and intercellular cell adhesion molecule-1 (ICAM-1) were detected in liver tissue.Pathological change was observed by optical microscope and electron microscopy.Results Tissue ATP decreased to less than 40％ of baseline after 30 min of warm ischemia,then restored to 70％ after 2 h of ECMO and returned to baseline after 4 h,while ATP of control group continued a further decline As compared with control group,SOD,GSH and HSP70 increased significantly in ECMO group (P＜0.05),while MDA and ICAM-1 decreased significantly (P＜0.05).Pathological changes of liver tissue observed by optical microscope and electron microscopy in ECMO group were significantly were significantly alleviated as compared with those in control group.Conclusion ECMO can supply oxygen and nutrients to liver after warm ischemia and increase energy reserve.By upregulating GSH,SOD and HSP-70 and other protective proteins,ECMO alleviates oxidative stress and liver damage ECMO also improves microcirculation and reduces neutrophil infiltration by protecting sinusoidal endothelial cells.

Objective To detect the protective effect of extracorporeal membrane oxygenation (ECMO) on Maastricht type Ⅱ donation after cardiac death (DCD) liver transplantation in pigs.Methods Twenty mini-pigs were randomized into ECMO group (n =10) and control group (n =10).Then 10 pigs in each group were randomized into donors and recipients.Maastricht type Ⅱ DCD model was induced in all of the 10 donors.Donors of ECMO group received 2-h ECMO after cardiac death,then underwent liver graft procurement.The donors of control group underwent liver procurement directly after cardiac death.Recipients of two groups underwent orthotopic liver transplantation without venovenous bypass.During this procedure,vital signs were monitored continuously,lactate and liver biochemistry were tested,and 5-day survival rate was observed.Results Maastricht type Ⅱ DCD model was successfully built in all of the donors with consequent dark liver.For donors of ECMO group,liver turned sanguinous and soft quickly after treatment of ECMO.There were no significant differences in operation time,anhepatic time and anhepatic hemodynamic changes between these two groups (P ＞ 0.05).As compared with control group,ECMO group had better hemodynamic parameters 30 min after reperfusion,lower lactate,ALT and AST levels 30 min after reperfusion and before closing the abdomen,and higher 5-day survival rate (P ＜ 0.05).Conclusion ECMO may improve the quality of Maastricht type Ⅱ DCD liver graft,and increase the survival rate of DCD liver transplantation.

Ebolavirus can cause hemorrhagic fever in humans with a mortality rate of 50%-90%. Currently, no approved vaccines and antiviral therapies are available. Human TIM1 is considered as an attachment factor for EBOV, enhancing viral infection through interaction with PS located on the viral envelope. However, reasons underlying the preferable usage of hTIM-1, but not other PS binding receptors by filovirus, remain unknown. We firstly demonstrated a direct interaction between hTIM-1 and EBOV GP in vitro and determined the crystal structures of the Ig V domains of hTIM-1 and hTIM-4. The binding region in hTIM-1 to EBOV GP was mapped by chimeras and mutation assays, which were designed based on structural analysis. Pseudovirion infection assays performed using hTIM-1 and its homologs as well as point mutants verified the location of the GP binding site and the importance of EBOV GP-hTIM-1 interaction in EBOV cellular entry.
Ebolavirus ; metabolism ; Flow Cytometry ; Glycoproteins ; metabolism ; Hepatitis A Virus Cellular Receptor 1 ; Hepatitis A Virus Cellular Receptor 2 ; Humans ; Membrane Glycoproteins ; metabolism ; Membrane Proteins ; metabolism ; Protein Binding ; Receptors, Virus ; metabolism ; Surface Plasmon Resonance ; Viral Envelope Proteins ; metabolism ; Viral Proteins ; metabolism

Coxsackievirus A16 belongs to the family Picornaviridae, and is a major agent of hand-foot-and-mouth disease that infects mostly children, and to date no vaccines or antiviral therapies are available. 2A protease of enterovirus is a nonstructural protein and possesses both self-cleavage activity and the ability to cleave the eukaryotic translation initiation factor 4G. Here we present the crystal structure of coxsackievirus A16 2A protease, which interestingly forms hexamers in crystal as well as in solution. This structure shows an open conformation, with its active site accessible, ready for substrate binding and cleavage activity. In conjunction with a previously reported "closed" state structure of human rhinovirus 2, we were able to develop a detailed hypothesis for the conformational conversion triggered by two "switcher" residues Glu88 and Tyr89 located within the bll2-cII loop. Substrate recognition assays revealed that amino acid residues P1', P2 and P4 are essential for substrate specificity, which was verified by our substrate binding model. In addition, we compared the in vitro cleavage efficiency of 2A proteases from coxsackievirus A16 and enterovirus 71 upon the same substrates by fluorescence resonance energy transfer (FRET), and observed higher protease activity of enterovirus 71 compared to that of coxsackievirus A16. In conclusion, our study shows an open conformation of coxsackievirus A16 2A protease and the underlying mechanisms for conformational conversion and substrate specificity. These new insights should facilitate the future rational design of efficient 2A protease inhibitors.
Coxsackievirus Infections ; virology ; Crystallography, X-Ray ; Cysteine Endopeptidases ; chemistry ; genetics ; Fluorescence Resonance Energy Transfer ; Hand, Foot and Mouth Disease ; enzymology ; pathology ; virology ; Humans ; Picornaviridae ; chemistry ; enzymology ; genetics ; Protein Conformation ; Structure-Activity Relationship ; Substrate Specificity ; Viral Proteins ; chemistry ; genetics

OBJECTIVE：To compare the c ontents of 6 kinds of ester alkaloids in raw aconite and 5 kinds of salt-processed products，and to provide reference for the optimization of salt-processing technology. METHODS ：Processed with bittern ， magnesium chloride ，sodium chloride ，calcium chloride and potassium chloride as excipients ，and HPLC method were adopted to determine the contents of 6 kinds of ester alkaloids in raw aconite and 5 kinds of salt-processed products. The effect/toxicity ratio and toxicity component index were used to evaluate the effect and toxicity of raw aconite and 5 kinds of salt-processed products. RESULTS： The linear range of benzoylneoaconitine ， benzoylhypoconitine， benzoylaconitine， neoaconitine， aconitine and hypoconitine were 2.440-24.40，2.240-22.40，2.020-20.20，2.780-27.80，2.240-22.40，2.240-22.40 μg/mL（r≥0.999 0）. RSDs of precision，stability and repeatability tests were all less than 2％. The recovery rates were 102.13％-104.87％（RSD＝0.98％，n＝ 6），100.00％-105.00％（RSD＝2.02％，n＝6），95.00％-99.29％（RSD＝1.77％，n＝6），100.41％-104.58％（RSD＝1.78％，n＝ 6），98.87％-99.90％（RSD＝0.41％，n＝6），100.20％-104.00％（RSD＝1.55，n＝6），respectively. The contents of total alkaloids in order from the largest to the smallest was as follows ：raw aconite ＞processed products of sodium chloride ＞processed products of bittern ＞processed products of potassium chloride ＞processed products of calcium chloride ＞processed products of magnesium chloride. The order of effect/toxicity ratio was processed products of potassium chloride ＞processed products of magnesium chloride＞processed products of sodium chloride ＞processed products of calcium chloride ＞processed products of bittern ＞raw aconite. The order of toxicity component index was raw aconite ＞processed products of sodium chloride ＞processed products of bittern＞processed products of calcium chloride ＞processed products of potassium chloride ＞processed products of magnesium chloride. CONCLUSIONS ：Using 5 kinds of salt as excipients ，the proce ssing technology has different degrees of “efficacy enhancing and toxicity reducing ”effect. Among them ，magne- sium chloride ，potassium chloride and calcium chloride are better for processing and can be used for processing aconite.