Objective:To synthesize Gd labeled probe targeting transporter protein(TSPO) 2-(8-amino-2-(4-chlorophenyl)imidazo[1, 2-a]pyridine-3-yl)- N, N-dipropylacetamide (CB86)-diethylene triamine pentaacetic acid (DTPA), and investigate its MRI effect in rheumatoid arthritis (RA) model. Methods:CB86-DTPA was prepared by coupling a bifunctional chelating agent, and then chelated with Gd to obtain MRI targeted contrast agent CB86-DTPA-Gd. The cytotoxicity, MR relaxation rate and in vitro stability of CB86-DTPA-Gd were determined. RA model was established with Freund′s adjuvant and the biodistribution study and MRI was performed. The RA lesion and its surrounding normal tissue were used as regions of interest (ROI) to calculate the signal to noise ratio (SNR). Independent-sample t test was used to analyze the data. Results:CB86-DTPA-Gd had excellent biosafety and a good MR relaxation rate ( r1=11.05 mmol·L -1·s -1). The survival rate of RAW264.7 cells and 4T1 cells was still more than 90% at the maximum concentration (20 μmol/L) of Gd 3+. CB86-DTPA-Gd also exhibited good stability in human serum and phosphate buffer saline solution (PBS; pH=7.4). The in vivo biodistribution showed that CB86-DTPA-Gd had better inflammatory targeting efficiency, and the uptake of Gd in the inflamed site of the ankle joint was still (2.33±0.29) percent dose rate per gram of tissue (%ID/g) at 120 min after injection. MicroMRI showed that the inflammation of the right ankle joint displayed significant enhancement after the injection of CB86-DTPA-Gd and Gd-DTPA. The SNR of CB86-DTPA-Gd group was up to 23.21±1.44, and the maximum intensification time was 90 min after injection, and can be significantly inhibited by CB86-DTPA at all time points ( t values: 6.083-12.451, all P<0.05), while the Gd-DTPA group had a strengthening time of 30 min after injection with the SNR of 16.12±1.24. Conclusion:CB86-DTPA-Gd shows good macrophage targeting and good uptake in arthritic reaction sites, and is expected to be a novel MRI molecular probe for peripheral inflammation imaging.

Objective To evaluate the efficacy and safety of 13-cis retinoid acid (13-CRA) and all trans retinoid acid (ATAR) redifferentiation therapy in patients with poorly differentiated thyroid cancer. Methods A single-center, randomized, double-blind, parallel controlled clinical trial was preformed. All patients were randomized into three groups. 78 cases were enrolled in each group. The patients were treated by 13-CRA in A group, by ATRA in B group, and by placebo in control group. The induced effects of retinoid acid (RA) and 131I treatment efficacies were defined as primary outcome of efficacy. Results After RA induction therapy, the effective rates in A, B, and control groups were 59.72%, 52.86% and 7.69%, respectively, with statistically significant difference among 3 groups (P<0.05). Compared with control group, A and B groups revealed significant induced efficacies (P<0.017), but there was no significant difference between A group and B group. After 131I treatment, the effective rates in A, B, and control group were 70.83%, 64.29%, and 28.21% respectively, with statistically significant difference (P<0.05). Compared with control group, the effective rates of 131I treatment in A and B groups were significantly raised (P<0.017), but there was no significant difference between A group and B group. The damage of skins and mucous membranes such as desquamation, dry skin, dry lips, dry eyes, etc occurred mostly in A group. The symptoms of nervous system such as headache, dizziness, etc occurred mostly in B group. Conclusions The induced differentiation of 13-CRA or ATRA is an effective method for the treatment of poorly differentiated thyroid carcinoma.

Objective To observe the effects of intervention of soothing liver and activating blood Chinese medicine on cardiac function and myocardial pathologic morphology of BMSCs transplanting on myocardial IRI of rats, and investigate its myocardial protection mechanism. Methods Model of myocardial IRI was established by coronary artery ligation in rats. SD rats were randomly divided into sham operation group, IRI group, BMSCs group and combined group. Rats in combined group were filled the stomach with soothing liver and activating blood Chinese medicine, and rats in other groups were filled the stomach with the same dose of normal saline. After 4 weeks, myocardial pathologic morphology was observed with light microscope. Cardiac function was detected with ultrasonic cardiogram.Results Compared with BMSCs group, heart function of the combined group improved, with significant statistical difference (P<0.05,P<0.01). Pathological observation showed that myocardial structure and pathological morphology were obviously promoted in the combined group.Conclusion Soothing liver and activating blood Chinese medicine could improve heart function and myocardial pathological morphology of IRI rats with BMSCs transplantation.

Objective To investigate the effects of soothing liver and activating blood Chinese medicine on myocardial cell apoptosis and related gene expression of BMSCs transplanting on myocardial ischemia reperfusion injury (IRI) of rats;To discuss its mechanism of protecting myocardium. Methods Model of myocardial IRI was established in rats. BMSCs were isolated, cultivated, and transplanted in IRI rats. SD rats were randomly divided into sham-operation group, IRI group, BMSCs group, and combined group. Rats in combined group received gavage with soothing liver and activating blood Chinese medicine, while rats in other groups received gavage with the same dose of normal saline. After 4 weeks, myocardial cell apoptosis, Bcl-2, and Bax protein expression in myocardial cells were detected by TUNEL method and immunohistochemical method. Results Compared with IRI group, myocardial cell apoptosis index in the combined group and BMSCs group was lower, Bax expression decreased, Bcl-2 expression significantly increased (P<0.01);Compared with BMSCs group, myocardial cell apoptosis index in the combined group was lower;Bax expression decreased, Bcl-2 expression increased (P<0.05, P<0.01). Conclusion Soothing liver and activating blood Chinese medicine can inhibit BMSCs transplantation in IRI rat myocardial cell apoptosis, promote myocardial regeneration, and protect myocardial cells.

Objective To study to take the oxidized Mannan?modified 786?0 in renal clear cell carcinom as tumor cells antigen to sensitize Dendrit?ic cells(DC)and to observe the its killing effect on renal clear cell carcinoma of CTLs induced. Methods Getting the peripheral blood mononucle?ar cells from the volunteers,and then to be stimulated to turn to be maturation by GM?CSF and IL?4 in vitro. Taking the clear renal carcinoma cell as the tumor antigen,and then making it to be modified by oxidized Mannan to acquire the tumor cell vaccine.Experimental groups include:blank group:DC?PBS group,control group:control?DC?786?0,experimental group:DC?Ox?Mannose?786?0 group. Taking the flow cytometry to detect the changes of DC phenotype,then taking the ELISA to detect the sencretion levels of supernatant of IL?12 of DC,then taking the CCK to detecte the cytotoxicity of lymphocytes(CTL)induced by DC of these experiment groups. Results Results by flow cytometry:the mature phenotype of DCs sensitized by Ox?Mannose?786?0 group included CD80,CD83,CD86 and HLA?DR expressed significantly higher than the other groups. As well as the secretion levels of IL?12. Meanwhile the cytotoxicity activity of lymphocytes(CTLs)induced by DCs which are sensitized by Ox?Mannose?786?0 increased more significantly than the other groups. Conclusion Glycosylated Antigens can be more effective in sensitizing antigen?presenting cells DC,and stimulating them to be maturation,while the killing effect to tumor cells also have noticeably improved.

Objective To synthesize 131I labeled anti-neuropilin-1 monoclonal antibody A6 (131IA6) and evaluate its biodistribution and imaging in malignant glioma xenografts.Methods (1) A6 was labeled with 131I by Iodogen method under the optimum labeling conditions,then the labeling efficiency,radiochemical purity and stability were measured in vitro.(2) In vitro bioactivity,cellular uptake and receptor affinity of 131I-A6 with U87MG cells were measured.(3) The nude mice bearing human U87MG cells were randomly divided into 5 groups with 5 in each group.The nude mice were sacrificed by cervical dislocation and dissected at 24,48,72,96,and 120 h,respectively,after intravenous injection of 1.2 MBq 131I-A6.The biodistribution of the agent was measured as ％ID/g,and the ratios of tumor/blood (T/B) and tumor/muscle (T/M) were calculated.(4) SPECT/CT imaging was performed in 6 mice including 3 in the competitive inhibition control group at 24,48,72,96,and 120 h post injection.Two-sample t test was used for data analysis.Results (1) The labeling yield of 131I-A6 was (95.46±3.34)％,and the radiochemical purity was more than 95％.At 96 h of incubation in PBS,the radiochemical purity was more than 85％.(2)131I-A6 had rapid accumulation in U87MG cells and reached the peak of (15.80±1.30)％ at 1 h.When the probe was incubated with large excesses of non-radioactive A6,the uptake level of 131I-A6 in U87MG cells was significantly inhibited (t=2.862,P＜0.05).Kd of 131I-A6 binding to NRP-1 was (1.67±0.14) nmol/L in U87MG cells.(3) Biodistribution study showed that the uptake in blood,liver and tumor was (8.00±1.42),(7.68±1.56) and (6.00±1.24) ％ID/g at 24 h,respectively.The uptake in muscle,brain and bone was lower.The T/B and T/M were 0.78±0.10 and 3.20±0.30 at 24 h,and they reached the highest level of 1.87±0.50 and 7.13±0.24 at 120 h.(4) The SPECT imaging showed that the tumors could be visualized at 24 h and delineated more clearly at 120 h post injection of 131I-A6.Conclusions 131I-A6 can be easily synthesized by Iodogen method with high radiochemical purity.The specific tumor uptake of 131I-A6,which correlates with NRP-1 expression in gliomas,suggests that it may be a new promising tumor targeting radiotracer.

Objective To study the feasibility of a novel probe 99Tcm-HYNIC-2(poly-(ethylene glycol),PEG) 4-Dimer (Dimer:E-[c (RGDfK) 2]) as a potential imaging agent for integrin αv β3 positive tumors,and also to observe the influence of an angiogenesis inhibitor,endostar,on the biodistribution and tumor uptake of the tracer in tumor bearing nude mice.Methods The expression of integrin αv β3 in human glioma cells U87MG was determined with immunofluorescence staining before and after treatment with endostar.99Tcm-HYNIC-2PEG4-Dimer was prepared and administered in U87MG tumor bearing mice in 6 h after either administration of endostar (200 μl) or saline (control group) and then biodistribution study was performed.Other 16 mice were divided into endostar treated group (20 mg/kg) and control group (saline) and then gamma imaging was performed in the two groups.Statistical significance of differences between the two groups was assessed using two-sample t test.Results Radiochemical purity of 99Tcm-HYNIC-2PEG4-Dimer was exceeded 95％.The expression of integrin αvβ3 in U87MG cell was high and gradually decreased after treatment with endostar.There was a negative dose-effect relationship between the dose of endostar and the expression of integrin αvβ3 with the peak effect at the dose of 400 μg/ml.The distribution study in vivo showed that the tracer uptake of U87MG tumors was high,but it decreased after injection of endostar.At 90 min,the ％ID/g of endostar and control groups were 1.50±0.08 and 6.27±0.33,respectively (t =40.23,P＜0.05).The average T/NT ratios of 99Tcm-HYNIC-2PEG4-Dimer uptake in the endostar and control groups were 1.02±0.11 and 2.58±0.36,respectively (t =10.25,P＜0.05).The integrin αv β3 positive expression ratios of tumor in endostar and control groups were (33.1 ±2.7) ％ and (81.5±3.2) ％,respectively (t =32.60,P＜0.05).Conclusions The novel probe 99Tcm-HYNIC-2PEG4-Dimer may be a promising radiotracer for integrin αvβ3-positive tumor imaging.It may be used for monitoring the therapeutic effect of endostar and may be potentially used for screening the candidates of anti-angiogenesis therapy.

Engineering and redesign of enzymes are important to industrial biocatalysis. Fusion enzyme technology, based on fusion protein design, is frequently used in multifunctional enzyme construction and enzyme proximity control. Here, we reviewed the recent progress in molecular design strategy and application studies of fusion enzymes. The concept and features of fusion enzymes were introduced, followed by a systematical summary of the design strategy of fusion enzymes. In particular, the effects of different linker properties on fusion enzymes and their possible mechanisms were discussed. In addition, recent studies on fusion enzyme applications were also discussed. Finally, based on our own studies on fusion enzymes and the current research progress, the key problems in fusion enzyme technology and perspectives of this field were discussed.
Biocatalysis ; Biotechnology ; Enzymes ; chemistry ; Protein Engineering ; Recombinant Fusion Proteins ; chemistry

Methanotrophs are a group of bacteria capable of utilizing methane as the sole carbon and energy source for their anabolism and catabolism. Since methanotrophs contain the unique enzymes of methane monooxygenases (MMOs), which can catalyze the oxidation of methane and short-chain alkanes and alkenes, they have potential applications in carbon recycle of nature and industrial biotechnology. Therefore, methanotrophs have been paid much more attention by the researchers in recent 20 years. In this paper, the latest progresses in studies of methanotrophs and MMOs were reviewed, including taxonomy, function and distribution of methanotrophs, and structure, function and genetic engineering of MMOs. The future research directions of methanotrophs and MMOs as well as their applications were also discussed.
Methane ; metabolism ; Methylococcaceae ; enzymology ; genetics ; Oxidation-Reduction ; Oxygenases ; metabolism

Methanotrophs use methane as the sole carbon and energy source, which cause slow growth, low cell density and hinder its industrial applications. One promising solution is to heterologously express methane monooxygenase (MMO) in other host cells that can be easily cultivated at high cell density. We systematically exploited the possibility of functional expression of pMMO by choosing different promoters and different host cells. The results showed that the recombinants could oxidize methane to methanol. In particular, ethanol could also be detected in the oxidized products, but the enzyme activity was instable, implying that some changes of pMMO expressed in the host cells might have occurred. In addition, SDS-PAGE analysis showed that many recombinants could express the subunits of pMMO, but the enzyme activity could not be detected. In conclusion, correct fold of pMMO in the host cells is important for its functional expression.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Enzymologic ; Genetic Vectors ; genetics ; Methane ; metabolism ; Methanococcaceae ; enzymology ; Methanol ; metabolism ; Oxygenases ; biosynthesis ; genetics ; Promoter Regions, Genetic ; Protein Folding ; Recombinant Proteins ; biosynthesis ; genetics