Objective To investigate the intracellular delivery of siRNAs through the applications of ultrasound targeted microbubbles destruction(UTMD)and biodegradable nanoparticles carriers.Methods Preparation of nanoparticles with and without RGD sequences,parameters optimization via L16(45)orthogonal design,control experiments in groups of optimization,RGD targeted nanoparticles,non-RGD nanoparticles and blank control, and determinations by inverted fluorescence microscope and flow cytometry were performed.Results The uptake and fluorescence intensity of PC-3 cells in group of RGD targeted nanoparticle was (93.49±1.37)% and 34.28±2.06 respectively,and that in group of optimization was (88.33±1.24)% and 30.59±3.93 respectively(P＞0.05).Whereas the uptake and fluorescence intensity of PC-3 cells in group of non-RGD nanoparticles was(71.24±2.80)% and 18.39±0.90 respectively,and that in group of optimization was (84.78±2.13)% and 27.18±0.91 respectively(P＜0.05).ConclusionsThe applications of UTMD with RGD targted nanoparticles cannot increase the intracellular delivery of siRNAs.

Objective To explore the difference of the contrast-enhanced ultrasound (CEUS) image and quantitative parameters between two different contrast agents Fluorocarbon and SonoVue. Methods The tumor model of colorectal carcinoma in nude mice was established by injecting CT26 cells into the subcutaneous space in hepatic area of 15 nude mice. CEUS was performed with Fluorocarbon and SonoVue on the 14th day after establishment. SonoLiver software was used to analyze the dynamic image quantitatively. The difference of the CEUS image and quantitative parameters between the two contrast agents was analyzed. Results Compared with normal liver parenchyma around the tumor, the tumor ultrasound contrast performance was fast forward and rewind with low enhancement. There was no signiifcant difference between the two kinds of microbubbles not only for CEUS image but also for quantitative parameters [maximum intensity (Imax):49.53%±24.38%vs 45.04%±17.03%, rise time (RT):11.68 s±3.07 s vs 13.76 s±2.92 s, time to peak (TTP):12.76 s±4.12 s vs 15.26 s±3.74 s, T1/2:50.57 s±28.32 s vs 48.75 s±9.85 s, Imax/TTP(V1):4.48±2.82 vs 3.18±1.49, (Imax-INT60)/(60-TTP)(V2):0.67±0.34 vs 0.60±0.20, AUC1:3032.78%±1343.12%vs 3258.77%±1369.84%, AUC2:11647.38%±6183.10%vs 10439.04%±4604.65%, AUC:14680.17%±7469.85%vs 13697.81%±5831.99%, Rate of AUC1:264.25±146.93 vs 222.24±92.16, Rate of AUC2:241.67±119.97 vs 231.97±100.34, all P>0.05). No mouse was dead during the CEUS examination. Conclusion Fluorocarbon had similar CEUS imaging effect and quantitative information compared with SonoVue.

Objective To explore the adeno-associated virus (AAV)gene transduction and cellular endocytosis mediated by ultrasound combined with microbubbles in two types of cells.Methods HeLa and NIH/3T3 cells were infected by rAAV2-EGFP at a concentration gradient to get the optimal concentrations for enhancement.At these concentrations,HeLa and NIH/3T3 cells were infected by rAAV2-EGFP mediated by ultrasound combined with microbubbles.The gene transduction efficiency were observed and measured by fluorescence microscopy and flow cytometry at 48 h after treatment.The cell viability was tested by CCK-8.The number and distribution of cellular clathrin-coated endocytic pits were observed by confocal fluorescence microscopy and transmission electron microscopy on 45 min after treatment.Results The optimal concentrations for HeLa and NIH/3T3 cells were 2000 v.g./cell and 10000 v.g./cell.Ultrasound combined with microbubbles significantly enhanced the transduction efficiency of rAAV2-EGFP (P <0.01) without significant cell viability decrease (P > 0.05 ).Confocal fluorescence microscopy and transmission electron microscopy demonstrated that clathrin-coated endocytic pits were more obviously increased in ultrasound combined with microbubbles mediated AAV transduction group than AAV transduction group. Conclusions Ultrasound combined with microbubbles can efficiently enhance the gene transduction of AAV,whose cellular transportation depends on cellular endocytosis,in two types of cells.Stimulating cellular endocytosis might be one of the mechanisms of enhanced cellular transportation of AAV mediated by ultrasound combined with microbubbles.

We reconstructed the erythromycin macrocyclic lactone (6-deoxyerythronolide B, 6dEB) synthesis pathway in Escherichia coli. We first cloned all the genes needed to synthesize the 6dEB into multi-gene co-expressed vectors. Then using the recognition sequences of isoschizomers Xba I/Spe I of vectors, we assembled the related genes into a series multiple-genes recombinant plasmids pBJ144, pBJ130. The recombinant plasmids pBJ144, pBJ130 were cotransformed into BAP1 to get the recombinant BAP1(pBJ144/pBJ130). SDS-PAGE analysis showed that individual genes were expressed correctly. After inducing at low temperature, adding propionate as substrate, we validated the crude product by mass spectrometry and the 6dEB yield was about 10 mg/L. These results indicated that the synthetic pathway of 6dEB was successfully assembled and reconstructed in Escherichia coli, which will greatly facilitate the reconstruction of whole erythromycin synthesis pathway and finally help to establish a stable research platform for developing of new derivatives of erythromycin and combinatorial biosynthesis of polyketide-type antibiotics.
Anti-Bacterial Agents ; biosynthesis ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Erythromycin ; analogs & derivatives ; biosynthesis ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics

Objective:To establish a mouse model of gastric cancer by inoculating MKN45 cells into mice with normal immune function utilizing microcarrier technology. Methods:A total of 60 male C57BL/6 mice were randomly divided into three groups, namely, 2D, con-trol, and 3D groups, according to the coculture system of MKN45 and microcarrier. The mouse models of gastric carcinoma were estab-lished by hypodermic injection. The time of tumorigenesis, rate of tumor formation, and pathological features were observed in each group. Results:In the 3D group, the time of tumor formation was short, whereas the rate of tumor formation was high (80%). No de-tectable tumor formations were observed in the 2D and control groups. HE and immunohistochemical staining of the transplantation tumor model showed evident characteristics of human gastric cancer. Conclusion:A human gastric cancer model in normal immune mice was successfully established. The onset and development mechanism of gastric cancer could be more effectively investigated in mice with normal immune function through this model. Moreover, a more valuable and new animal model for the research and devel-opment of anticancer drug was established.

ObjectiveTo investigate the effect of intraperitoneal transplantation of human liver-derived stem cells in the prevention and treatment of alcoholic fatty liver disease in mice. Methods A total of 30 male C57BL/6 mice were randomly divided into blank control group (group N), model control group (group M), and stem cell transplantation group (group S). The mice in group N were fed a normal diet, and those in the other two groups were fed Lieber-DeCarli alcohol liquid diet; at the same time, the mice in group S were given intraperitoneal transplantation of human liver-derived stem cells twice a week. After six weeks of intervention, body weight and liver index were measured, and the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were also measured. The levels of TG and non-esterified fatty acid (NEFA) in the liver were measured, and liver pathological examination and oil red O staining of the liver were performed. One-way ANOVA was used for comparison of continuous data between multiple groups, and the SNK-q test was used for further comparison between two groups. Results There were significant differences in the serum levels of ALT, AST, TG, TC, and HDL-C and the content of TG and NEFA in the liver between the three groups (F=66.94, 7.15, 8.02, 18.64, 386, 2314 and 3049, all P＜0.05), Compared with group N, group M showed significant increases in levels of ALT, AST, and TG in serum and levels of TG and NEFA in liver tissue (all P＜0.05). Group S had significantly lower levels of ALT, AST, and TG in serum and levels of TG and NEFA in liver tissue than group M (all P＜0.05). Liver HE staining and oil red O staining showed that group S had a significantly lower degree of liver steatosis than group M. ConclusionIntraperitoneal transplantation of human liver-derived stem cells has a marked effect in the prevention and treatment of alcoholic fatty liver disease in mice.

Objective:To evaluate the qualitative diagnosis of contrast enhanced ultrasound (CEUS) in different sizes′ benign and malignant breast nodules (BNs) of BI-RADS 4.Methods:A total of 506 BNs in 467 patients from Shanghai General Hospital between October 2017 and May 2019 classified as BI-RADS 4 were divided into large size group (>20 mm, n=198) and small size group (≤20 mm, n=308) according to their largest diameter lines. The CEUS characteristics of benign and malignant BNs in two groups were analyzed by Chi-square test. The CEUS characteristics of BNs were assigned scores for diagnosis of benign and malignant BNs with different sizes, the diagnostic efficacy of these scores was evaluated by ROC curve. Results:There were statistic differences in the degree, size change of the enhanced lesions, enhancement time of lesions compared with surrounding tissues, radial enhancement or not, homogeneity of enhancement, with or without penetrating of perfusion between benign and malignant BNs in both groups (all P<0.001). In small size group, with the critical value of 2.5 points, area under curve(AUC) of ROC curve was 0.793, and the sensitivity, specificity, accuracy of CEUS was 82.67%, 60.22% and 69.48%, respectively. In large size group, with the critical value of 3.5 points, AUC of ROC curve was 0.901, and the sensitivity, specificity, accuracy of CEUS was 85.42%, 80.00% and 82.65%, respectively. Conclusions:CEUS has great significance in qualitative diagnosis of benign and malignant in BI-RADS 4 BNs with different sizes. The biopsy could be avoided in BNs greater than 20 mm, while biopsy is still needed in BNs less than or equal to 20 mm.

The medical magnetic resonance (MR) image reconstruction is one of the key technologies in the field of magnetic resonance imaging (MRI). The compressed sensing (CS) theory indicates that the image can be reconstructed accurately from highly undersampled measurements by using the sparsity of the MR image. However, how to improve the image reconstruction quality by employing more sparse priors of the image becomes a crucial issue for MRI. In this paper, an adaptive image reconstruction model fusing the double dictionary learning is proposed by exploiting sparse priors of the MR image in the image domain and transform domain. The double sparse model which combines synthesis sparse model with sparse transform model is applied to the CS MR image reconstruction according to the complementarity of synthesis sparse and sparse transform model. Making full use of the two sparse priors of the image under the synthesis dictionary and transform dictionary learning, the proposed model is tackled in stages by the iterative alternating minimization algorithm. The solution procedure needs to utilize the synthesis and transform K-singular value decomposition (K-SVD) algorithms. Compared with the existing MRI models, the experimental results show that the proposed model can more efficiently improve the quality of the image reconstruction, and has faster convergence speed and better robustness to noise.