Development of highly efficient electrocompetent cells for electroporation of Geobacillus thermoglucosidasius NCIMB 11955 / 生物工程学报

Geobacillus thermoglucosidasius is a kind of Gram-positive facultative anaerobic bacteria. The fast growth rate under high temperature and less susceptibility to microbial contamination enable G. thermoglucosidasius to be a desirable producer of biofuels and high-value-added chemicals for the next-generation industrial biotechnology. However, compared with the classical model strain Escherichia coli, the applications of G. thermoglucosidasius are hampered by its low transformation efficiency. This study aimed at obtaining competent cells with high transformation efficiency through inactivating restriction enzymes, adding cell membrane inhibitors and cell wall weakening agents. The results showed that the electro-transformation efficiency achieved 1.2×104 CFU/(μg DNA) by knocking out four genes encoding restriction enzymes. Adding a certain amount of tween 80, dl-threonine and glycine further increased the competent efficiency about 22.5, 44, and 334 times, respectively. The electro-transformation efficiency was enhanced to 4.6×106 CFU/(μg DNA) under the optimized conditions, laying a foundation for genetic manipulation and metabolic engineering of G. thermoglucosidasius.

Automatic synthesis and myocardial imaging of 11C-meta-hydroxyephedrine / 中华核医学与分子影像杂志

Objective:To realize a fully automated synthesis of 11C-meta-hydroxyephedrine (mHED) and to perform imaging studies with it. Methods:11C-mHED was prepared by the 11CH 3-triflate method. The crude product was purified by semi-preparative high performance liquid chromatograph (HPLC) to obtain the final product. The radiochemical purity and specific activity were determined by radio-HPLC. The myocardial uptake and excretion process of the agent were monitored by microPET/CT imaging on 5 normal SD rats. The clinical imaging value was evaluated using PET/CT imaging in a patient (male, 42 years old) with myocardial infarction. Results:The automated synthesis of 11C-mHED was realized by a commercial synthesizer. The total synthesis time was about 30 min. The radiochemical yield was (15±2)% (non-decay corrected, n=10) and the radiochemical purity was greater than 98%. The specific activity was about 65 GBq/mmol. MicroPET/CT imaging in normal SD rats showed the myocardial uptake was highest at 10 min after the injection of imaging agent, and then the imaging agent was gradually excreted from the myocardium through the liver and gallbladder. PET/CT imaging of a patient with myocardial infarction showed an imaging agent defect near the apex in the inferior wall of the left ventricle, which was matched with results of ultrasound and electrocardiogram examination. Conclusions:11C-mHED can be successfully prepared automatically, with high radiochemical yield and specific activity. It can also highly concentrate in the myocardium, and the imaging effect with this agent is good in a patient with myocardial infarction.

Preliminary study on 99Tc m-3PRGD 2 imaging to verify the anti-angiogenesis mechanism and efficacy of Mongolian medicine Sendeng-4 decoction for rheumatoid arthritis / 中华核医学与分子影像杂志

Objective:To explore the therapeutic mechanism of Mongolian medicine Sendeng-4 decoction for rheumatoid arthritis by 99Tc m-hydrazinonicotinamide-(polyethylene glycol) 4-E[(polyethylene glycol) 4-c((Arg-Gly-Asp)fk)] 2 (3PRGD 2) imaging. Methods:A total of 200 female SD rats (age: 6-7 weeks) were divided into collagen-induced arthritis (CIA) group ( n=176) and blank control group ( n=24). Rats in the CIA group were divided into Sendeng-4 decoction treatment group ( n=24), etanercept treatment group ( n=24), and negative control group ( n=24) by simple random sampling method. 99Tc m-3PRGD 2 SPECT/CT imaging was performed before and after modeling and treatment. The differences of target/non-target (T/NT) ratio and serological, pathological, and immunohistochemical results among groups were compared by one-way analysis of variance or Kruskal-Wallis rank sum test. The correlation was analyzed by Pearson correlation or Spearman correlation analysis. Results:There were 95 (95/176) CIA models successfully established. The T/NT ratios of Sendeng-4 decoction treatment group and etanercept treatment group were lower than that of negative control group (0.260± 0.094, 0.238±0.099, 0.766±0.144 ; F=163.00, P<0.001), while there was no significant difference between the two drug treatment groups ( P>0.05). After drug treatment, serum levels of vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α) and α vβ 3 were significantly lower than those of negative control group ( F values: 49.43-92.36, all P<0.001), pathological score was also lower than that of negative control group ( H=34.25, P<0.001), and levels of immunohistochemical makers (VEGF, TNF-α, α vβ 3, CD31, CD34) were also lower than those of negative control group ( H values: 13.51-26.84, all P<0.001), while there were no significant differences between the two drug treatment groups (all P>0.05). The T/NT ratios were positively correlated with above indictors in Sendeng-4 decoction treatment group ( r values: 0.56-0.59, rs values: 0.49-0.69), etanercept treatment group ( r values: 0.50-0.55, rs values: 0.46-0.70) and negative control group ( r values: 0.55-0.80, rs values: 0.58-0.86, P<0.001 or P<0.05). Conclusion:Verified by 99Tc m-3PRGD 2 SPECT/CT imaging and molecular pathology, Mongolian medicine Sendeng-4 decoction can inhibit neovascularization by down-regulating vascular factors such as VEGF, resulting in delaying the progression of the disease and improving clinical symptoms.

Experimental study of SPECT myocardial blood flow quantitative analysis in animals with coronary microvascular disease / 中华核医学与分子影像杂志

Objective:To compare the quantitative parameters of myocardial blood flow based on SPECT imaging and those determined by PET imaging in coronary microvascular disease (CMVD) animal models, in order to clarify the accuracy and feasibility of SPECT quantitative analysis in CMVD.Methods:Seven Saanen milk goats (either male or female; (20±5) kg), were selected for establishing CMVD animal models by microsphere embolization. Dynamic myocardial perfusion imaging (DMPI) with one-day method of resting + ATP stress 99Tc m-methoxyisobutylisonitrile (MIBI) SPECT was performed before and after the modeling, respectively. One-day method of resting + ATP stress 13N-ammonia PET DMPI was performed after the modeling. The quantitative parameters determined by SPECT and PET after the modeling, including stress myocardial blood flow (SMBF), resting myocardial blood flow (RMBF) and myocardial flow reserve (MFR), were compared by paired t test. Parameters based on SPECT after modeling were compared with those of baseline levels. Bland-Altman analysis was applied to access the agreement between SPECT and PET. Results:Four of the seven experimental goats were fully imaged. The RMBF(ml·g -1·min -1; 1.52±0.27 vs 1.29±0.20), SMBF(ml·g -1·min -1; 0.74±0.19 vs 0.99±0.26), and MFR (0.53±0.16 vs 0.76±0.10) of the left ventricle (global) obtained by SPECT and PET in CMVD models were not significantly different ( t values: 3.121, 1.195, 1.930, all P>0.05). Among left anterior descending branch (LAD), left circumflex (LCX) and right coronary artery (RCA), the RMBF, SMBF and MFR values quantified by SPECT and PET were neither statistically significant ( t values: 0.182-2.734, all P>0.05). Bland-Altman analysis showed the quantitative parameters measured by SPECT and PET DMPI in left ventricle, LAD, LCX, RCA had a good consistency. The difference between the two methods for determining RMBF was up to 0.63 ml·g -1·min -1, and that of SMBF was up to 0.66 ml·g -1·min -1. All points are within the 95% confidence limit; MFR differs at most by 0.56, and 14/16 points were within 95% confidence limit. The RMBF (ml·g -1·min -1) of left ventricle measured by SPECT after modeling was not significantly different from that before modeling (1.52±0.27 vs 1.57±0.36; t=0.166, P>0.05); the SMBF (ml·g -1·min -1) and MFR after modeling were significantly lower than those before modeling (0.74±0.19 vs 2.34±0.89, 0.53±0.16 vs 1.39±0.31, t values: 3.836, 6.309, both P<0.05). Similar results were found when comparing the parameters of LAD/LCX/RCA after modeling with those before modeling (RMBF t values: 0.191, 0.235, 0.195, all P>0.05; SMBF/MFR t values: 0.411-19.911, all P<0.05). Conclusion:The blood flow quantitative parameters measured by SPECT imaging have a good consistency with those based on PET imaging, and the myocardial blood flow quantitative analysis of SPECT can evaluate the blood flow perfusion of CMVD.

Micro PET/CT molecular imaging observation on mechanism of anti-myocardial ischemia/reperfusion injury action of / 中国医学影像技术

Objective: To explore the mechanism of anti-myocardial ischemia/reperfusion injury (MIRI) action of Guangzao Qiwei pills in rats using micro PET/CT molecular imaging. Methods: Totally 120 male SD rats were randomly divided into high dose group, medium dose group and low dose group of Guangzao Qiwei pills, as well as blank control group, model control group and positive control group (each n=20). Micro PET/CT imaging was performed before and 24 h, 48 h, 72 h after model establishment. Volume of interest (VOI) and the mean standardized uptake value (SUVmean) in ischemic region at each time point were calculated. Serum oxidative stress parameters (superoxide dismutase [SOD] and malondialdehyde [MDA]) and marker enzymes of myocardial injury (lactate dehydrogenase [LDH] and creatine kinase [CK]) were observed. All rats were sacrificed after the ending of imaging, and then myocardial pathological changes were observed. Results: Different degrees of imaging agent sparsity-defect were observed in each dose group 24 h and 48 h later. The imaging agent sparsity was still observed in low dose group which recovered in medium and high dose groups. SUVmean of ischemic region gradually increased with time in all 3 dose groups and metabolism gradually recovered (F=121.82, 450.82, 435.75, all P<0.05). Each dose group can Free radicals were eliminated and the damage of myocardial injury reduced in all 3 dose groups, better effect on SOD, on MDA and LDH or on CK were observed in the high dose, the medium dose and the low dose group, respectively (all P<0.05). Conclusion: The mechanism of anti-MIRI action of Guangzao Qiwei Pills achieve by eliminating free radicals in the body, reducing oxidative stress injury and affecting the activities of relevant enzymes. All dose Guangzao Qiwei Pills can protect rats from MIRI, high dose has the best effect. PET/CT molecular imaging allows non-invasive, in vivo and dynamic detection of MIRI and accurately evaluation on the protective role of Guangzao Qiwei Pills in MIRI.

Experimental study of 99Tcm-3PRGD2 imaging on the angiogenesis of rheumatoid arthritis / 中华核医学与分子影像杂志

Objective@#To observe the uptake of 99Tcm-3 polyethylene glycol Arg-Gly-Asp dimer (3PRGD2) in rat models of rheumatoid arthritis (RA), in order to provide theoretical foundation for early diagnosis of RA.@*Methods@#The healthy female SD rats were divided into collagen induced arthritis (CIA) group (n=100), osteoarthritis (OA) group (n=20) and control group (n=20). Bovine collagen type Ⅱ emulsion was used for arthritis induction to establish CIA models. OA models were established by injection of L-cysteine and papain. Gamma imaging was performed before and 15 d, 30 d after the model establishment. The mediastinum was selected as non-target (NT) area and the target (T)/NT ratios were calculated. The serum levels of vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), αVβ3 before and after the model establishment were measured. Pathological and immunohistochemical detection were performed. One-way analysis of variance, Pearson and Spearman correlation analyses were used to analyze the data.@*Results@#There were 58 CIA models successfully established. Before the model establishment, T/NT ratios of CIA group, OA group and control group were 0.215±0.049, 0.210±0.050, 0.209±0.051, respectively (F=0.093, P>0.05). The T/NT ratios of the above three groups were 0.405±0.230, 0.223±0.045, 0.211±0.049 (F=12.601, P<0.05) 15 d post-model establishment, and those were 0.572±0.182, 0.238±0.045, 0.212±0.055 (F=65.147, P<0.05) 30 d post-model establishment. T/NT ratios of CIA group were positively correlated with the levels of serum VEGF, TNF-α, αVβ3, pathological score and the levels of immunohistochemical markers (VEGF, TNF-α, αVβ3, CD31, CD34; r values: 0.391-0.721, rs values: 0.365-0.669, all P<0.05).@*Conclusion@#99Tcm-3PRGD2 can be specifically up-taken by RA lesions in rat models, thus has the potential in early synovial neovascularization imaging of RA.

Cardiac neuroceptor imaging on acute myocardial ischemia model using 11C-methyldopamine / 中华核医学与分子影像杂志

Objective To evaluate 11C-MDA,a cardiac sympathetic nerve presynaptic molecular probe,in acute myocardial ischemia (AMI) and to compare with 13N-Ammonia myocardial perfusion imaging.Methods Twelve Bama miniature pigs were used to establish AMI models.11C-MDA and 13N-Ammonia PET/CT were performed before and after model establishment.The defect fraction,ratio of defect to normal region,defect volume of 11C-MDA and 13N-Ammonia PET/CT were calculated.Paired-t test and twosample t test were used.Results Eight models were established successfully and 4 pigs died.The defect fractions on 11C-MDA and 13N-Ammonia myocardial imaging were significantly different (13.4±3.3 vs 7.4±1.0,18.8±4.4 vs 4.8±1.0,10.5±4.2 vs 4.4±0.9;t values:4.901,8.864,4.030,all P＜0.001) at the first,third and sixth month post-model establishment.The ratios of defect to normal region on 11C-MDA and 13N-Ammonia PET/CT myocardial images were significantly different (0.47±0.14 vs 0.59±0.10,0.43±0.13 vs 0.61±0.09;t values:-2.166,-3.415,both P＜0.05) at the first and third month post-model establishment.The defect volumes on 11C-MDA and 13N-Ammonia PET/CT were significantly different ((4.20±0.34) vs (2.55±0.11) cm3,(10.66±0.71) vs (2.46±0.12) cm3,(5.95±0.50) vs (2.44±0.11) cm3;t values:12.925,32.149,19.440,all P＜0.001) at the first,third and sixth month post-model establishment.Conclusions Myocardial ischemia-reperfusion and cardiac sympathetic restoration might occur at different times.The recovery of myocardial ischemia-reperfusion is earlier than that of the cardiac sympathetic nerve.

The value of recurrence or metastasis of 18F-FDG PET/CT and serumCEA in patients with advanced gastric cancer after neoadjuvant chemotherapy combined with surgical treatment / 国际检验医学杂志

Objective To investigate the value of 18F-FDG PET/CT and serum CEA in patients with advanced gastric cancer after neoadjuvant chemotherapy combined with surgical treatment.Methods 96 cases of patients with advanced gastric cancer who underwent neoadjuvant chemotherapy combined with surgery as the research object,after more than 2 years of follow-up,PET/CT imaging and serum CEA levels were detected,and the diagnostic value of recurrence or metastasis of the two groups were evaluated and analyzed.Results Of the 96 patients enrolled in the study,there were 47 cases of recurrence and metastasis,PET/CT combined with CEA for recurrence or metastasis positive diagnosis rate and negative diagnosis rate was significantly higher than that of pure PET/CT and serum CEA,the difference was statistically significant(P<0.05).The sensitivity,specificity and accuracy of PET/CT combined with CEA in the diagnosis of recurrence or metastasis were higher than that of PET/CT and serum CEA,the difference was statistically significant(P<0.05).Conclusion The combined monitoring of 18F-FDG PET/CT imaging and serum CEA level in patients with advanced gastric cancer after neoadjuvant chemotherapy combined with surgical treatment is helpful to improve the diagnosis of metastasis or recurrence.

Advances in imaging methods for early diagnosis of rheumatoid arthritis / 中华核医学与分子影像杂志

Rheumatoid arthritis (RA) is a systemic autoimmune disease with unknown etiology.The main feature of RA is chronic,symmetrical and invasive arthritis.RA may present with severe joint deformity and functional loss,which has higher disability and fatality rates.At present,no effective measures can reverse bone damage associated with RA clinically.Therefore,early diagnosis of RA is critical to improve prognosis.In recent years,imaging increasingly plays an important role in the early diagnosis of RA.This article reviews recent advances in imaging methods for early diagnosis of RA.

The Effect of Norcantharidin on Human Rectal Cancer Colo 320 Cells / 昆明医科大学学报

Objective To investigate the effect of norcantharidin on growth inhibition and induction of apoptosis of human rectal cancer Colo 320 cells. Methods Norcantharidin (NCTD) in different concentrations were added to rectal cancer Colo 320 cells. Morphological characteristics of apoptosis were observed using the light microscope and transmission electron microscope. The expressions of Bag-1 and Bcl-2 proteins were tested by Western blotting. The growth inhibition of Colo 320 cells on the cell cycle was observed by flow cytometry. Results The apoptosis morphological changes of Colo 320 cells were observed by the light microscope and transmission electron microscopy. Flow cytometry analysis showed that the cell count of G2/M phase in experimental group was higher than that in control group ( <0.05) but the cell counts of G0/G1 and S phases have decreased in experimental group after treatment with NCTD at the concentrations of 5μg/mL, 10μg/mL and 20 μg/mL, and presented dosage dependence relations. The expressions of Bag-1 and Bcl-2 proteins have decreased. Conclusion Norcantharidin has inhibitory effect on rectal cancer Colo 320 cells, and the effect may be related to the cell cycle arrest and apoptosis.