Objective To isolate and identify the potential binding partners of LRRK2,a gene linked to both dominant familial form and sporadic form of Parkinson's disease,thus to further our knowledge of its function.Methods We used a sequence containing full-length of COR domain and part of ROC and MAPKKK domain as bait.The bait amplified by polymerase chain reaction(PCR) was then cloned into a yeast expression plasmid pGBKT7.After being sequenced and analyzed,pGBKT7-bait was transformed into the yeast strain AH109.Western blot was performed to confirm the expression of pGBKT7-bait in AH109 yeast strain.Then human fetal brain cDNA library was trarnsformed into that yeast strain.which could express pGBKT7-bait fusion protein.The yeast strain which contained pGBKT7-bait and human fetal brain cDNA library was plated on quadruple dropout medium (SD/-Trp/-Leu/-His/-Ade)containing X-a-gal.We retested these positive colonies using 2 independent yeast strains AH109 contained pGBKT7-bait or pGBKT7,respectively.At last,these plasmids isolated from these true positive colonies were analyzed by bioinformatics.Results We obtained 9 true positive colonies,these colonies were sequenced, and we performed sequence Blast in GenBank.Three colonies of the 9 positive colonies were not in open reading-frames.Among other 6 colonies,there were known proteins including spermatid perinuclear RNA-binding protein(STRBP)and BCL2-associated athanogene 5 isoform b(BAG5),as well as unknown proteins including tyrosine phosphatase non-receptor type(PTPN23),1(3)mbt-like 3 isoform b(L3 MBTL3),RALY RNA binding protein-like isoform 1(RALYL),and Homo sapiens mRNA for KIAA1783 protein,partial cds(KIAA 1783).Conclusion True positive colonies of LRRK2 are successfully obtained by the yeast 2-hybrid.Our screened proteins may provide a new research clue for revealing biological functions of LRRK2,pathogenesis of Parkinson's disease,and other neurodegerations.

OBJECTIVETo investigate the effect of TiO₂nanotube arrays covalently modified by recombinant human bone morphogenetic protein- 2(rhBMP- 2) on the early bioactivity of mesenchymal stem cells (MSC) in vitro and to provide experimental evidence for the biochemical modification of titanium implants.

METHODSIn the experiment group, double titanium nanotube arrays were prepared by anodization, and were chemically grafted with rhBMP- 2. Mechanically polished pure titanium was used as blank control group, and titanium dioxide nanotubes was used as negative control A group, and titanium dioxide nanotubes + carbonyldiimidazole as negative control B group. Field emission scanning electron microscope (FE- SEM) and X- ray photoelectron spectroscopy (XPS) were used to detect the morphology and physicochemical properties of the experiment group, blank control group and the negative control group. Cell adhesion on the specimen surface of the experiment group, blank control group and negative control group on the 1st day was tested. Cell proliferation on the 1st, 3rd and 5th day and alkaline phosphatase activity on the 5th, 7th and 11th day was also tested.

RESULTSFE- SEM showed that the surface of titanium nanotubes loaded with rhBMP- 2 possessed visible miliary particulate matter. XPS showed that nitrogen peak in the group of titanium nanotubes loaded with rhBMP-2 was significantly greater that those in the other groups. FE- SEM showed that the cells on the surface of the experimental group on the 1st day spread well, better than those in the control group and negative control group. Cell proliferation activity on the 1st day in different groups was not obvious (P>0.05), the A value of the experimental group on the 3rd and 5th day (3.295 ± 0.153, 3.823 ± 0.059) were significantly higher than those in the control group (2.479 ± 0.064, 3.131 ± 0.096) and negative control A group (2.715 ± 0.075, 3.371 ± 0.047) and negative control B group (2.756 ± 0.132, 3.637 ± 0.047) (P<0.05). Alkaline phosphatase activity on the 5th, 7th and 11th day in the experimental group (0.0477 ± 0.0287, 0.0615 ± 0.0016, 0.0667 ± 0.0018) were better than those in the control group, negative control A group and negative control B group (P<0.05).

CONCLUSIONSTitanium nanotube arrays can be loaded with rhBMP-2 by biochemical methods and have good biocompatibility.

Alkaline Phosphatase ; metabolism ; Bone Morphogenetic Protein 2 ; pharmacology ; Cell Adhesion ; Cell Proliferation ; Humans ; Materials Testing ; Mesenchymal Stromal Cells ; Microscopy, Electron, Scanning ; Nanotubes ; chemistry ; ultrastructure ; Photoelectron Spectroscopy ; Recombinant Proteins ; pharmacology ; Surface Properties ; Titanium ; chemistry ; Transforming Growth Factor beta ; pharmacology

Objective:To explore the therapeutic effect of multi-mode sequential combination of artificial liver in the treatment of hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF).Methods:The clinical data of HBV-ACLF patients treated with artificial liver in Wuxi Fifth People′s Hospital from January 2018 to June 2021 were retrospectively analyzed. Eighty-six patients were divided into artificial liver multi-mode sequential combination therapy group (sequential combination group) and conventional treatment group. The cytokine level changes and model for end-stage liver disease (MELD) score were analyzed at 14 days of disease duration. The survival outcome and complications of artificial liver were analyzed after 30 days of follow-up. Two independent samples t test and chi-square test were used for statistical analysis. Cox regression analysis was used to analyze the risk factors of death, and Kaplan-Meier method was used to analyze the survival rate of patients. Results:A total of 86 patients were enrolled, including 48 patients in sequential combination group with the average number of artificial liver of 4.68 times/person, and 38 patients in conventional treatment group with the average number of artificial liver of 3.17 times/person. At 14 days of disease duration, interleukin (IL)-6, IL-8, interferon γ-inducible protein (IP)-10 level and MELD score in sequential combination group decreased significantly than those in the conventional treatment group ( t=3.80, 3.62, 4.95 and 1.11, respectively, all P<0.050). After 30 days of follow-up, 63 patients survived and 23 patients died. Cox regression analysis showed that baseline international normalized ratio (hazard ratio ( HR)=0.558, 95% confidence interval ( CI) 0.193 to 0.856, P=0.027), baseline antithrombin Ⅲ activity ( HR=0.876, 95% CI 0.824 to 0.932, P<0.001), artificial liver mode ( HR=0.819, 95% CI 0.236 to 0.992, P=0.005), spontaneous peritonitis ( HR=0.170, 95% CI 0.045 to 0.647, P=0.009) and hepatic encephalopathy ( HR=0.004, 95% CI 0.001 to 0.030, P<0.001) were independent influencing factors for 30-day survival outcome. The cumulative survival rate of sequential combination group was higher than that of conventional treatment group, and the difference was statistically significant ( χ2=5.45, P=0.020). There were no significant differences in the proportions of bleeding, deep vein thrombosis, heart rate and blood pressure instability between the two groups ( χ2=0.63, 1.20 and 0.54, respectively, all P>0.050). The platelet decline of patients in sequential combination group was slighter than that in conventional treatment group, and the difference was statistically significant ( t=-4.17, P=0.002). Conclusions:Multi-mode sequential combination therapy of artificial liver could eliminate cytokines and reduce MELD score more effectively in patients with HBV-ACLF, and prolong the survival time of patients and have little effect on platelet count.

【Objective】 To explore the precautions of pre-transfusion examination in patients with antibodies to erythrocyte protective solution, discrepant ABO blood typing results, and positive unexpected antibodies, so as to ensure the safety of blood transfusion. 【Methods】 The screen cells were divided into two groups according to the presence or absence of washing reagent red blood cells in normal saline. One group had untreated forward typing cells, antibody screening cells and identification panel, and the other group had saline-washed reverse typing cells, antibody screening cells and identification panel. The experiments were carried out by microcolumn gel method, saline medium method and polyamine method to analyze the effect of red blood cell preservation solution on serum agglutination reaction of specific patients. 【Results】 Among the 8 patients, forward typing was AB (+ ) in 1 patient, B (+ ) in 4, and A(+ ) in 3, and the reverse typing were interfered. The plasma of 8 patients agglutinated with unwashed reverse typing cells (saline tube method), screen cells and identification panels (saline tube method plus cassette method), while not agglutinated with the polybrene method. The interference was eliminated as using washed reverse typing cells (salinetube method), screen cells and identification panels (saline tube method plus cassette method). 【Conclusion】 The erythrocyte preservation solution affected patients’ blood group typing, but not affected the outcome of blood transfusion as no adverse reactions occurred.

Humans ; Warfarin/therapeutic use* ; Cytochrome P-450 CYP2C9/genetics* ; Anticoagulants/therapeutic use* ; Genotype ; Heart Valves ; China ; Apolipoproteins E/genetics* ; Dose-Response Relationship, Drug ; Receptors, Calcitriol/genetics*

Objective: To investigate the influence of different discontinuation time of aspirin and clopidogrel before off-pump coronary artery bypass grafting (OPCABG) on postoperative bleeding and blood products transfusion requirement. Methods: Three hundred and fifty-three coronary artery disease patients who underwent OPCABG from January 2017 to January 2018 at Department of Cardiac Surgery, Zhongshan Hospital, Fudan University were retrospectively analysed. There were 268 males and 85 females, aged (66.0±9.1)years. All patients were divided into three groups: (1) guideline-recommended group: patients who discontinued clopidogrel for >5 days without discontinuing aspirin before surgery; (2) without discontinuing group: patients who discontinued clopidogrel for ≤5 days without discontinuing aspirin before surgery; (3) discontinuing group: patients who discontinued clopidogrel for >5 days with discontinuing aspirin before surgery. Postoperative bleeding recorded as chest tube drainage (CTD) volume and blood products transfusion requirement and perioperative complications were recorded. CTD volumes within 12 hours after surgery between groups were compared by Mann-Whitney U tests, CTD volumes after 12 hours postoperatively were compared by repeated measures analysis of variance and blood products transfusion and complications incidence were compared by χ² test or Fisher′s precise test. Results: The 12 hours CTD volumes of guideline-recommended group, without discontinuing group, discontinuing group after surgery were 280(153) ml (M(Q_R)), 291(229) ml, 225(161) ml, respectively. There were no significant differences in postoperative 12 hours CTD volumes (P=0.865), red blood cells transfusion incidence (χ²=2.626, P=0.149) and fresh frozen plasma (FFP) transfusion incidence (χ²=1.258, P=0.324) between guideline-recommended group and without discontinuing group. However, the 12 hours CTD volumes were significantly higher in guideline-recommended group patients compared with disconutinuing group patients (U=5 247, P=0.002). No significant differences were observed in red blood cells (χ²=0.182, P=0.757) and FFP (χ²=0.083, P=0.839) transfusion rate between these two groups. Repeated measures analysis of variance indicated that when patients began to take antiplatelet drugs (aspirin and clopidogrel) after 12 hours postoperatively, the change of CTD volumes beyond 12 hours after surgery didn′t differ either between guideline-recommended group and without discontinuing group (F=0.019, P=0.941) or between guideline-recommended group and discontinuing group (F=2.447,P=0.113). Besides, the incidence of perioperative arrhythmia was significantly higher in guideline-recommended group patients compared with without discontinuing group patients (4.8% vs. 0, χ²=5.073, P=0.038). Conclusions OPCABG patients who discontinued aspirin before surgery had lower postoperative 12 hours CTD volumes but similar blood products transfusion rate and CTD volumes beyond 12 hours postoperatively compared with patients adhering to the current guideline-recommended protocol. And for patients who discontinued clopidogrel for ≤5 days, postoperative CTD volumes and blood products transfusion requirement were similar but the incidence of perioperative arrhythmia was significantly lower compared with guideline-treated patients.

The theory of "combination of five meridians" focuses on meridians and acupoints as well as compatibility and interrelationship of generation and restriction of five elements. By regulating corresponding meridians to diseased, the relationship of unbalancedis balanced, which can regulate body function and effectively treat diseases. Under the guide of theory "the combination of five meridians", according to etiology and pathogenesis of post-stroke urinary incontinence, wind, fire and phlegm, from three aspects of deficiency, excess and deficient root and excessive surface, under the treatment principle of "nourishing the mother if it is deficient, reducing the son if it is excessive", the original meridian, son meridian, mother meridian, conquering meridian and conquered meridian are selected while the methods of nourishing the mother, reducing the son, reinforcing the suppression and developing the weak are applied, so as to discuss the acupuncture treatment for post-stroke urinary incontinence, which can provide new clinical thoughts of acupuncture treatment for post-stroke urinary incontinence.