1H-NMR and MS Based Metabolomics Study of the Intervention Effect of Curcumin on Hyperlipidemia Mice Induced by High-Fat Diet

Curcumin, a principle bioactive component of Curcuma longa L, is well known for its anti-hyperlipidemia effect. However, no holistic metabolic information of curcumin on hyperlipidemia models has been revealed, which may provide us an insight into the underlying mechanism. In the present work, NMR and MS based metabolomics was conducted to investigate the intervention effect of curcumin on hyperlipidemia mice induced by high-fat diet (HFD) feeding for 12 weeks. The HFD induced animals were orally administered with curcumin (40, 80 mg/kg) or lovastatin (30 mg/kg, positive control) once a day during the inducing period. Serum biochemistry assay of TC, TG, LDL-c, and HDL-c was conducted and proved that treatment of curcumin or lovastatin can significantly improve the lipid profiles. Subsequently, metabolomics analysis was carried out for urine samples. Orthogonal Partial Least Squares-Discriminant analysis (OPLS-DA) was employed to investigate the anti-hyperlipidemia effect of curcumin and to detect related potential biomarkers. Totally, 35 biomarkers were identified, including 31 by NMR and nine by MS (five by both). It turned out that curcumin treatment can partially recover the metabolism disorders induced by HFD, with the following metabolic pathways involved: TCA cycle, glycolysis and gluconeogenesis, synthesis of ketone bodies and cholesterol, ketogenesis of branched chain amino acid, choline metabolism, and fatty acid metabolism. Besides, NMR and MS based metabolomics proved to be powerful tools in investigating pharmacodynamics effect of natural products and underlying mechanisms.     

Genome engineering of Agrobacterium tumefaciens using the lambda Red recombination system

Agrobacterium tumefaciens has been widely used as a tool for transgenesis in plants. The availability of its genome sequence should facilitate the directed engineering of improved properties; however, the current genome engineering options are laborious. Here, we investigated whether the lambda R ed operon can be applied for recombineering of the A. tumefaciens genome. First, we built an expression plasmid for A. tumefaciens employing a tetracycline-inducible promoter to regulate the Red operon. This multicopy plasmid was then itself modified in A. tumefaciens to verify that the Red operon was functional. Then, we modified the endogenous A. tumefaciens tumor-inducing plasmid and the linear chromosome. These results extend recombineering technology to a new host and indicate a fast and convenient way to engineer the A. tumefaciens genome for functional genomics and strain improvements.     

RT-PCR heteroduplex analysis permits differentiation of transgene and host gene expression in a transgenic animal model

In transgenic animal models, the conservation of DNA sequences between the transgene and the host wild-type gene can complicate the evaluation of the expression of each gene. The potential for gene silencing may complicate matters further. Here we report the use of RT-PCR heteroduplex analysis to differentiate the expression of a transgene and its homologous wild-type, even when these genes are very similar in their respective DNA sequences. We designed RT-PCR primers to amplify identically sized 243-bp fragments within the DNA binding domain of the p53 gene from both human and mouse mRNA samples. Ten samples from human p53 (273H) transgenic mice and 10 samples from wild-type controls were tested. Heteroduplex bands were formed in all transgenic samples but were absent from all wild-type samples. In addition, RT-PCR heteroduplex analysis was able in one sample to differentiate a silenced transgene from its wild-type allele, without the assistance of sequencing or labeling. In summary, the RT-PCR heteroduplex analysis is easy to use and has the ability to screen a large number of samples in a short time. The RT-PCR heteroduplex analysis is especially useful for the detection of expression when a transgene and the host homologous endogenous allele are too conserved in sequence to design species-specific RT-PCR primers.     

云南禄丰古猿地点的松鼠类化石

记述了云南禄丰晚中新世石灰坝古猿地点发现的松鼠科化石。禄丰石灰坝动物群的松鼠动物共有 7属 7种 :地松鼠Sciurotamiaswangisp .nov .,树松鼠Tamiopssp .、Callosciurussp .、Dremomysprimitivussp .nov .和飞松鼠Miopetauristaasiaticasp .nov .、Hylopetodondianensegen .etsp.nov.、Pteromyinaegen.etsp .indet.。该动物群松鼠类动物的组成以树松鼠和飞松鼠为主 ,反映了较为湿润的热带—亚热带森林环境。在动物地理上 ,其成员几乎局限于东南亚分布 ,具有浓厚的现代东洋界色彩     

Association between variants of EXT2 and type 2 diabetes: a replication and meta-analysis

Recent publications have found an association between variants of exostosin 2 (EXT2) gene and the risk of type 2 diabetes in some population but not in others. In an attempt to address these inconsistencies, we investigated EXT2 variants in two independent cohorts, and conducted a literature-based meta-analysis. Through regression model, we assessed the relationship between the EXT2 single nucleotide polymorphisms (SNPs) (rs3740878, rs11037909 and rs1113132) and the risk of type 2 diabetes in Han Chinese population, including a total of 2,533 cases and 2,643 controls. We combined our data with that from previously published studies and performed a meta-analysis to evaluate the effect size of the gene. Consistent with some studies, we found marginal association for the rs3740878 (OR = 1.07, 95 % CI = 0.99, 1.16, p = 0.09), rs11037909 (OR = 1.07, 95 % CI = 0.99, 1.16, p = 0.08), and rs1113132 (OR = 1.08, 95 % CI = 1.00, 1.17, p = 0.06) in our 2 cohorts. Meta-analysis, comprising 9,224 type 2 diabetes and 10,484 controls, revealed that three SNPs (rs3740878, rs11037909 and rs1113132) in EXT2 were significantly associated with type 2 diabetes (ORs range from 1.06 to 1.07, p = 0.038, p = 0.008 and p = 0.005, respectively). Variation in the EXT2 locus appears to be associated with a small increase in the risk of type 2 diabetes. However, well-designed prospective studies with larger sample size and more ethnic groups are needed to further validate the results.     

Mesenchymal Stem Cell Transplantation Enhancement in Myocardial Infarction Rat Model under Ultrasound Combined with Nitric Oxide Microbubbles

Objective

This study evaluated the effects of ultrasound combined with the homemade nitric oxide (NO) micro-bubble destruction on the in vitro proliferation, apoptosis, and migration of mesenchymal stem cells (MSCs). Furthermore, we studied whether or not irradiation of the NO micro-bubble combined with bone-marrow derived MSC infusion had a better effect on treating myocardial infarction. The possible mechanism of MSC delivery into the infarcted myocardium was also investigated.

Methods

The murine bone marrow-derived MSCs were isolated, cultured, irradiated, and combined with different concentrations of NO microbubbles. MTT proliferation assay, annexin V-FITC apoptosis detection, migration assay, and RT-PCR were performed 24 h after the irradiation. The NO micro-bubbles was a intravenously injected, followed by the infusion of MSCs, which were labeled by CM-Dil. Myocardium was harvested 48 h later and the distribution of MSCs was observed by laser scanning confocal microscope after frozen sectioning. Echocardiography, histological examination, RT-PCR, and western blotting were performed four weeks after the cell transplantation.

Results

Ultrasound combined with 1:70 NO micro-bubbles had no significant impact on the proliferation or apoptosis of MSCs. Transwell chamber findings demonstrated that MSCs migrated more efficiently in group that underwent ultrasound combined with 1:70 NO micro-bubbles. The Real-time PCR results indicated that the expression of CXCR4 was much higher in the group undergoing ultrasound combined with 1:70 NO micro-bubbles. The normalized fluorescence intensity greatly increased in the group of US+NO micro-bubbles and the cardiac function was also markedly improved. Immunohistochemical staining showed that the capillary density was much greater in the group of US+NO micro-bubbles as compared to that of the other groups. RT-PCR and western blotting also revealed a higher SDF-1 and VEGF expression in the group of US+NO micro-bubbles.

Conclusions

NO micro-bubbles could be used in the cell transplantation, which efficiently promoted the MSC homing into the infarcted myocardium.     

Improving simvastatin bioconversion in Escherichia coli by deletion of bioH

Simvastatin is an important cholesterol lowering compound and is currently synthesized from the natural product lovastatin via multistep chemical synthesis. We have previously reported the use of an Escherichia coli strain BL21(DE3)/pAW31 as the host for whole-cell biocatalytic conversion of monacolin J acid to simvastatin acid. During fermentation and bioconversion, unknown E. coli enzyme(s) hydrolyzed the membrane permeable thioester substrate dimethylbutyryl-S-methyl mercaptopropionate (DMB-S-MMP) to the free acid, significantly decreased the efficiencies of the whole-cell bioconversion and the downstream purification steps. Using the Keio K-12 Singe-Gene Knockout collection, we identified BioH as the sole enzyme responsible for the observed substrate hydrolysis. Purification and reconstitution of E. coli BioH activity in vitro confirmed its function. BioH catalyzed the rapid hydrolysis of DMB-S-MMP with kcat and Km values of 260+/-45 s(-1) and 229+/-26 microM, respectively. This is in agreement with previous reports that BioH can function as a carboxylesterase towards fatty acid esters. YT2, which is a delta bioH mutant of BL21(DE3), did not hydrolyze DMB-S-MMP during prolonged fermentation and was used as an alternative host for whole-cell biocatalysis. The rate of simvastatin acid synthesis in YT2 was significantly faster than in BL21(DE3) and 99% conversion of 15 mM simvastatin acid in less than 12 h was achieved. Furthermore, the engineered host required significantly less DMB-S-MMP to be added to accomplish complete conversion. Finally, simvastatin acid synthesized using YT2 can be readily purified from fermentation broth and no additional steps to remove the hydrolyzed dimethylbutyryl-S-mercaptopropionic acid is required. Together, the proteomic and metabolic engineering approaches render the whole-cell biocatalytic process more robust and economically attractive.