Objective To study the clinical value of three-dimensional vascular reconstruction of power Doppler imaging in monitering acute rejection of renal transplant.Methods Eighty-eight renal allograft patients were studied by three-dimensional vascular reconstruction and graded the vascular flow.Results The ratio of successful renal transplant three-dimensional vascular reconstruction reached 95.5% in 88 cases.Blood flow gradation was 4-5 levels in 30 controls and 0-4 levels in 58 patients with acute renal transplant rejection. Blood flow gradation was closely relevant to the time when the blood flow began to improve and the renal function began to restore after treatment, value of r being -0.9224 and -0.9223 respectively (P

A sensitive electrochemical biosensor based on Aflatoxin-Oxidase (AFO) was developed for detection of sterigmatocystin (ST). The enzyme was immobilized on chitosan-single-walled carbon nanotubes (CS-SWCNTs) hybrid film, which attached to the poly-o-phenylenediamine (POPD)-modified Au electrode. The fabricated procedures of the biosensor were characterized with atomic force microscopy (AFM), fourier transform-infrared spectroscopy (FT-IR), and electrochemical impedance spectroscopy (EIS). The cyclic voltammetric results of the biosensor indicated that AFO exhibited a surface-controlled and quasi-reversible electrochemical redox behavior with a formal potential of -0.436 V (vs. Ag/AgCl) in 0.1 mol/L PBS (pH 7.0), which resulted from the direct electron transfer between entrapped AFO and the underlying electrode. The enzymatic electrode exhibited an excellent electrocatalytic response to ST. The linear range of ST determination was from 10 ng/mL to 310 ng/mL with correlation coefficient of 0.997, the detection limit was 3 ng/mL (S/N=3), and the response time was less than 10 seconds. The apparent Michaelis-Menten constant (K(M)app) was estimated to be 7.13 micromol/L. The biosensor had the advantages of good repeatability and stability, remaining 85.6% of its original current value after storage at 4 degrees C for a month, and the RSD for 11 replicate determination of 20 ng/mL ST was 3.9%. This AFO/CS-SWCNTs/POPD/Au modified electrode showed high selectivity and sensitivity in real sample analysis, giving values of recovery in the range of 87.6%-105.5%. The proposed method can be applied to the determination of ST in real samples with satisfactory results.
Aflatoxins ; Biosensing Techniques ; methods ; Chitosan ; chemistry ; Electrons ; Nanotubes, Carbon ; Oxidation-Reduction ; Oxidoreductases ; Phenylenediamines ; chemistry ; Sterigmatocystin ; analysis

We has studied the feasibility of preventing protein from denature during covalent immobilization by "conformation memory", which was achieved by freeze-drying under enzyme active conformation and cross-linked with carrier under micro-aqueous media (MAM). Horseradish peroxidase (HRP) and chitosan beads have been used as the model enzyme and carrier. The MAM consisted of 99% dioxane and 1% water. We compared the immobilized HRP under MAM with that under traditional aqueous solvent, found that the optimum temperature of both was raised to 60 degrees C, and the optimum pH was 6.5. However, the MAM-immobilized HRP had shown less activity loss during usage and six times higher activity than that immobilized under aqueous solvent. After 30 min incubation at 70 degrees C, the MAM-immobilized HRP remained 75.42% activity while the aqueous-media-immobilized enzyme only 15.4%. The MAM-immobilized HRP has shown a better operation stability with 77.69% residue activity after 5 times of repeat operation while the aqueous-media-immobilized enzyme only 16.67%. In addition, the MAM-immobilized HRP had also shown more advantages when used in phenol removal. We constructed enzyme electrodes (CS-HRP-SWCNTs/Au) to further display the different properties of the two immobilized HRP. MAM-immobilized HRP-electrode has shown two times stronger response signal to H2O2 than that immobilized under aqueous media, which indicated a better enzyme activity of MAM-immobilized HRP. Our research demonstrated that the conformation memory, to some extent, did contribute to preventing protein from denaturing when use HRP as a model, and it is feasible to immobilize enzyme by covalent cross-linking method under micro-aqueous media.
Chitosan ; chemistry ; Enzyme Stability ; Enzymes, Immobilized ; metabolism ; Freeze Drying ; Horseradish Peroxidase ; chemistry ; metabolism ; Protein Conformation ; Solvents

Background Traditional Chinese medicine (TCM), especially herbal medicine, has been widely used in China and now is also being increasingly used in other countries for the treatment of cardiovascular diseases. Although many studies have demonstrated that certain Chinese herbal products are effective and safe for the treatment of cardiovascular diseases, most of these lack sufficient quality. Therefore, large randomized clinical trials and further scientific research to determine its safety, effectiveness are necessary.QiShen YiQi Dripping Pills (QSYQDP) is a herbal preparation clinically used in the treatment and prevention of coronary artery disease. Preliminary observations have shown its safety and effectiveness. Methods/Design This randomized, controlled trial will recruit 3600 patients with a history of myocardial infarction. Patients will be randomized into two groups by a Centr-Randomized System. One group receives QSYQDP, the other group receive aspirin. This trial protocol will describe eligibility criteria, detailed information on the treatment definition, blinding, endpoints, statistical methods, sample size determination, data management, legal aspects, and the current status of the trial. Discussion This trial is one of the first randomized, controlled clinical trial to evaluate the efficacy and safety of traditional Chinese herbal medicine in the treatment and secondary prevention of coronary artery disease. The results of this study should help to define the role of TCM in modern medical care, as well as to provide the management strategy for CAD patients in China and other countries.

Objective To analyze the correlation between the clinical characteristics and the incidence of postoperative hepatic encephalopathy in patients with cirrhosis who received transjugular intrahepatic portosystemic shunt ( TIPS) . Methods The clinical data of 80 patients with liver cirrhosis hospitalized in the Fifth Medical Center of PLA General Hospital from March 2015 to March 2017 were retrospectively analyzed. The related factors of hepatic encephalopathy complications after TIPS treatment were analyzed. Results The cumulative incidence of hepatic encephalopathy was about 23. 75%(19／80) in one year after TIPS operation. Through single factor analysis and multi factor Logistic regression analysis,it was found that the two factors of preoperative platelet level and the location of tips blood shunt were closely related to the occurrence of hepatic encephalopathy after TIPS operation. The ROC curve suggested that the AUC of preoperative platelet level and the location of tips blood shunt were 0. 657 and 0. 668, respectively. The sensitivity and specificity of the former were 70. 0%,52. 6% and the cutoff value was 51 ×109／L, respectively, while those of the latter were 70. 5% and 63. 2%, respectively. Conclusion Preoperative platelets level＞51×109／L or shunting of the left branches of the portal vein may indicate a lower incidence of hepatic encephalopathy.

Objective To investigate the effect of autophagy on acquired secondary resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) in patients of non-small cell lung cancers (NSCLC). Methods Ninety-eight patients with pathological confirmation of lung adenocarcinoma were selected, and they were treated with EGFR-TKI. Among them, 49 patients were sensitive to EGFR-TKI, (EGFR-TKI sensitive group), and the others were resistant (EGFR-TKI resistant group). The expressions of mTOR, Beclin-1 and LC3 were detected by immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot method. Results The immunohistochemistry results showed that the Beclin-1 and LC3 positive expression rates of tumor tissue in EGFR-TKI resistant group were significantly lower than those in EGFR-TKI sensitive group:24.49%(12/49) vs. 83.67%(41/49) and 22.45% (11/49) vs. 85.71% (42/49), and the mTOR positive expression rate was significantly higher than that in EGFR-TKI sensitive group: 77.55% (38/49) vs. 20.41% (10/49), and there were statistical differences (P<0.05). The RT-PCR results showed that the Beclin-1 and LC3 positive expressions of tumor tissue in EGFR-TKI resistant group were significantly lower than those in EGFR-TKI sensitive group: 0.723 ± 0.029 vs. 2.542 ± 0.104 and 0.886 ± 0.034 vs. 2.234 ± 0.164, and the mTOR positive expression was significantly higher than that in EGFR-TKI sensitive group:2.142 ± 0.132 vs. 0.638 ± 0.031, and there were statistical differences (P<0.05). The Western blot results showed that the Beclin-1 and LC3 positive expressions of tumor tissue in EGFR-TKI resistant group were significantly lower than those in EGFR-TKI sensitive group:0.315 ± 0.037 vs. 1.226 ± 0.017 and 0.420 ± 0.016 vs. 1.023 ± 0.014, and the mTOR positive expression was significantly higher than that in EGFR-TKI sensitive group: 0.986 ± 0.032 vs. 0.282 ± 0.021, and there were statistical differences (P<0.05). In EGFR-TKI sensitive group and EGFR-TKI resistant group, the Beclin-1 and LC3 showed positive correlation, and the Beclin1 and LC3 showed negative correlation with mTOR. Conclusions The signaling molecules of autophagy play an important role in secondary resistance to EGFR-TKI in patients of NSCLC. As a regulation mechanism of autophagy, mTOR takes part in the procedure of resistance to EGFR-TKI, and give a new biological marker of the predication of drug resistance. Meanwhile, it gives a new target of drug resistance reversal and individualized treatment.

Objective:To explore the role and mechanism of kidney brain protein (KIBRA) down-regulation in cognitive dysfunction caused by chronic cerebral hypoperfusion.Methods:Ninety male SPF grade Sprague Dawley (SD) rats were divided into four groups according to random number table: sham operation group ( n=15), chronic hypoperfusion group (2VO group, n=25), chronic hypoperfusion stereotaxic injection of AAV-KIBRA group (2VO+ AAV-KIBRA group, n=25), chronic hypoperfusion stereotaxic injection of AAV-Vector group (2VO+ AAV-vector group, n=25). Chronic cerebral hypoperfusion model was established by bilateral ligation of common carotid artery, and stereotactic injection of 2 μL AAV-KIBRA or AAV-vector was performed for 30 days.Morris water maze, in vitro electrophysiology, p21-activated kinase 3(PAK3) activity detection, Western blot, immunoprecipitation and Golgi staining were used to detect spatial learning and memory ability, long-term potentiation(LTP), KIBRA level expression, PAK3 activity changes and the distribution of dendritic spines.SPSS 16.0 statistical software was used for statistical data.One-way ANOVA was used to compare the differences between groups.LSD test was used to compare the significance of data differences between the two groups.Welch test was used for uneven variance. Results:After 1 month of chronic cerebral hypoperfusion, the level of KIBRA in the hippocampus of rats was detected by homogenate and Western blot, and it was found that the level of KIBRA in 2VO group was lower than that of sham group(73.49±4.12)% ( P<0.01). AAV-KIBRA injection in hippocampal CA1 region significantly up-regulated the level of KIBRA to (91.91±7.01)% over 2VO group ( P<0.01). Morris water maze test showed that the latency of the 2VO group(3rd-7th day trail data: (48.18±2.82)s, (43.45±2.27)s, (32.27±2.22)s, (26.55±2.37)s, (17.18±2.67)s) were significantly longer than those of the sham group((41.67±2.74)s, (32.58±2.57)s, (22.50±2.94)s, (16.91±2.39)s, (8.75±1.52)s) (all P<0.05), and the latencies of the 2VO+ AAV-KIBRA group 3rd-7th day trail data: (43.83±2.95)s, (35.25±2.15)s, (26.58±2.03)s, (19.92±2.17)s, (17.75±1.35)s) was significantly shorter than that of the 2VO group ((all P<0.01). The Morris water maze test with the platform removed showed that the latency of rats in the 2VO group to reach the platform region was significantly longer than that of the sham group, while the latency of rats in the 2VO+ AAV-KIBRA group to reach the platform region was significantly shorter than that in the 2VO group ( P<0.01). At the same time, the retention time and the crossing times in the platform region of 2VO group were less than those of the sham group ( P<0.01), but the retention time and the crossing times in the platform region of 2VO+ AAV-KIBRA group were significantly higher than those in the 2VO group ( P<0.01). The electrophysiological records of the brain slices showed that the relative excitatory postsynaptic field potential of 2VO group (1.43±7.43) was significantly lower than that of sham group (2.21±6.54) after high frequency stimulation, while the relative excitatory postsynaptic field potential of 2VO+ AAV-KIBRA group (1.90±8.15) was higher than that of 2VO group ( P<0.01). Immunoprecipitation in rat hippocampus revealed that PAK3 could be detected by Western blot assay when KIBRA was precipitated.The results showed that the relative enzyme activity of PAK3 in 2VO hippocampal tissue (0.64±0.04) was significantly lower than that in sham group (1.02±0.07), while the relative enzyme activity of PAK3 in 2VO+ AAV-KIBRA group (0.86±0.03) was significantly higher than that in 2VO group.Golgi staining showed that the density of dendritic spines in 2VO hippocampal neurons((6.85±0.43)/10 μm) was significantly lower than that in sham group((11.83±0.58)/10 μm), while the density of dendritic spines in 2VO+ AAV-KIBRA group((10.22±0.39)/10 μm) was significantly higher than that in 2VO group. Conclusion:The down-regulated of KIBRA after chronic cerebral hypoperfusion plays a key role in cognitive dysfunction and is also involved in the decrease of synaptic functional plasticity.The downregulation of KIBRA is involved in the structural plasticity of dendrites through the regulation of PAK3 activity.Therefore, KIBRA may be an important target for the prevention and treatment of cognitive function of chronic cerebral hypoperfusion.

The aim of the study is to obtain an efficient expression of recombinant ubiquitin-like specific protease 1 (Ulp1) by gene engineering. We cloned the Ulp1p, active fragment (403 aa-621 aa) of Ulp1, from Saccharomyces cerevisia, and subcloned into pGEX/Rosetta (DE3) to form an expression plasmid, pGEX-Ulp1p-His6. In order to enhance the solubility of GST-Ulp1p-His6, we purified the fusion protein GST-Ulp1p-His6 by either glutathione S-transferase agarose or Ni-NTA resin chromatography, the purity was up to 98%. We utilized the protein to cleave the SUMO fusions, and the specific activity of GST-Ulp1p-His6 was 1.375 x 10(4) U/mg. This study showed that the recombinant protein GST-Ulp1p-His6 displayed high specificity and activity.
Cloning, Molecular ; Cysteine Endopeptidases ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Fungal Proteins ; biosynthesis ; genetics ; Glutathione Transferase ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; enzymology ; Solubility

OBJECTIVETo review kidney transplantation in the center and analyze the risk factors affecting long-term allograft survival.

METHODSThirty-two relative variables were analyzed with SAS statistical software. Using Log-rank method, we investigated influence of these variables on short-and long-term survival of grafts. Kaplan-Meier analysis was used to estimate the 1-, 3-, 5-, 10-years graft survival rates and half-life. Proportional hazards regression analysis (Cox model) was used to assess and rank the relative risk of potential variables.

RESULTSThe 1-, 3-, 5-, 10-years graft survival rates were 83%, 75%, 66% and 48%. After excluding the patients died with functioning grafts, the 1-, 3-, 5-, 10 years grafts survival rate increased to 89%, 82%, 75% and 69%, respectively. The mean half-life was 8.78 +/- 0.14 and 14.09 +/- 0.20 years, respectively. By Log-rank analysis, factors affecting short- and long-term graft survival were identified as: renal function, duration of graft function became normal, cold-ischemia time, presence of acute rejection, delayed graft function, immunosuppressive regimen, complication, infection, anti-rejection therapy. Cox model multivariate analysis showed that there were 18 factors affecting graft survival.

CONCLUSIONSNew immunosuppressive agents not only significantly increase short-term graft survival, but also have the better long-term outcome tendency. Making assurance to get high quality donor organ and minimizing the death with graft function may be the most feasible way to prolong graft survival at present.

Adult ; Cadaver ; Female ; Graft Survival ; drug effects ; Humans ; Immunosuppressive Agents ; pharmacology ; Kidney Transplantation ; Male ; Multivariate Analysis ; Transplantation, Homologous

Objective To investigate the effect of early continuous renal replacement therapy on the prognosis of urinary sepsis after surgery for upper urinary tract calculi.Methods The clinical data of 59 patients with urinary sepsis after surgery for upper urinary tract calculi were retrospectively analyzed.In 59 patients with urinary sepsis,9 patients with early persistent renal replacement therapy were selected as the experimental group and 50 patients without early continuous renal replacement therapy as the control group. The age,sex ratio,diabetes,hypertension,the average operation time were no significant difference(P>0.05).Preoperative urinary tract infection,the experimental group preoperative urinary tract infection was significantly higher than the control group(77.8% vs.32.0%,P<0.05).In the results of preoperative urine culture positive ratio,the experimental group was also significantly higher than the control group,the difference was statistically significant(P<0.05).The changes of prognostic factors in patients with or without continuous renal replacement therapy were analyzed.Results In terms of the changes of postoperative laboratory results,the decrease of procalcitonin(PCT),C-reactive protein,white blood cell count and neutrophil ratio in the experimental group was significantly larger than that in the control group, and the difference was statistically significant;on the increase of hemoglobin,platelet count,fibrinogen,the standard bicarbonate level from blood gas analysis in the experimental group increased significantly more than the control group,the difference between the two groups were statistically significant.Conclusions Early renal replacement therapy in patients with urinary sepsis after upper urinary tract calculi surgeries can improve the prognosis of patients.