Drug target discovery by magnetic nanoparticles coupled mass spectrometry / 药物分析学报

Drug target discovery is the basis of drug screening.It elucidates the cause of disease and the mechanism of drug action,which is the essential of drug innovation.Target discovery performed in biological sys-tems is complicated as proteins are in low abundance and endogenous compounds may interfere with drug binding.Therefore,methods to track drug-target interactions in biological matrices are urgently required.In this work,a Fe3O4 nanoparticle-based approach was developed for drug-target screening in biofluids.A known ligand-protein complex was selected as a principle-to-proof example to validate the feasibility.After incubation in cell lysates,ligand-modified Fe3O4 nanoparticles bound to the target protein and formed complexes that were separated from the lysates by a magnet for further analysis.The large surface-to-volume ratio of the nanoparticles provides more active sites for the modification of chemical drugs.It enhances the opportunity for ligand-protein interactions,which is beneficial for capturing target proteins,especially for those with low abundance.Additionally,a one-step magnetic separation simplifies the pre-processing of ligand-protein complexes,so it effectively reduces the endogenous interference.Therefore,the present nanoparticle-based approach has the potential to be used for drug target screening in biological systems.

Identification and functional analysis of soybean stearoyl-ACP Δ⁹ desaturase (GmSAD) gene family / 生物工程学报

Stearoyl-ACP Δ⁹ desaturase (SAD) catalyzes the synthesis of monounsaturated oleic acid or palmitoleic acid in plastids. SAD is the key enzyme to control the ratio of saturated fatty acids to unsaturated fatty acids in plant cells. In order to analyze the regulation mechanism of soybean oleic acid synthesis, soybean (Glycine max) GmSAD family members were genome-wide identified, and their conserved functional domains and physicochemical properties were also analyzed by bioinformatics tools. The spatiotemporal expression profile of each member of GmSADs was detected by qRT-PCR. The expression vectors of GmSAD5 were constructed. The enzyme activity and biological function of GmSAD5 were examined by Agrobacterium-mediated transient expression in Nicotiana tabacum leaves and genetic transformation of oleic acid-deficient yeast (Saccharomyces cerevisiae) mutant BY4389. Results show that the soybean genome contains five GmSAD family members, all encoding an enzyme protein with diiron center and two conservative histidine enrichment motifs (EENRHG and DEKRHE) specific to SAD enzymes. The active enzyme protein was predicted as a homodimer. Phylogenetic analysis indicated that five GmSADs were divided into two subgroups, which were closely related to AtSSI2 and AtSAD6, respectively. The expression profiles of GmSAD members were significantly different in soybean roots, stems, leaves, flowers, and seeds at different developmental stages. Among them, GmSAD5 expressed highly in the middle and late stages of developmental seeds, which coincided with the oil accumulation period. Transient expression of GmSAD5 in tobacco leaves increased the oleic acid and total oil content in leaf tissue by 5.56% and 2.73%, respectively, while stearic acid content was reduced by 2.46%. Functional complementation assay in defective yeast strain BY4389 demonstrated that overexpression of GmSAD5 was able to restore the synthesis of monounsaturated oleic acid, resulting in high oil accumulation. Taken together, soybean GmSAD5 has strong selectivity to stearic acid substrates and can efficiently catalyze the biosynthesis of monounsaturated oleic acid. It lays the foundation for the study of soybean seed oleic acid and total oil accumulation mechanism, providing an excellent target for genetic improvement of oil quality in soybean.

Seed-specific expression of heterologous gene DGAT1 increase soybean seed oil content and nutritional quality / 生物工程学报

Enhancing soybean (Glycine max) oil production is crucial to meet the market demand of vegetable oil. Diacylglycerol acyltransferase (DGAT) catalyzes the final acylation reaction of triacylglycerol (TAG) synthesis, acting as one of the rate-limiting enzymes for oil biosynthesis in plant seeds. Here, a cDNA clone VgDGAT1A encoding the DGAT1 protein was isolated from the high oil plant Vernonia galamensis. VgDGAT1A was specifically overexpressed in soybean seeds, and several high-generation transgenic lines (T7) were obtained by continuous selection. qPCR analysis showed that VgDGAT1A was highly expressed in the mid-development stage (30-45 DAF) of the transgenic seeds. Accordingly, the DGAT enzyme activity in the transgenic seeds was increased by 7.8 folds in comparison with the wild-type controls. Seed oil and starch contents were, respectively, increased by 5.1% (Dry weight) and reduced by 2%-3% in the transgenic soybeans. Importantly, protein content was not significantly different between transgenic and control seeds. Seed weight and germination rate of the transgenic lines exhibited no negative effect. Fatty acid profiling demonstrated that antioxidant oleic acid (C18:1Δ9) content in the transgenic seed oil was elevated by 8.2% compared to the control, and correspondingly, easily-oxidized linoleic acid (C18:2Δ9,12) and linolenic acid (C18:3Δ9,12,15) were decreased by 6% and 2% respectively. Taken together, seed-specific overexpression of an exogenous VgDGAT1A gene can break the negative linkage of oil and protein contents in soybean seeds, indicating that engineering of this highly-active DGAT enzyme is an effective strategy to improve oil yield and nutritional value in oilseeds.

Using journal as the carrier to improve medical students' scientific research ability / 中华医学科研管理杂志

Cultivating students' creative spirit was an important aim of higher education,and improving scientific research ability was an effective way to realize this aim.Using Journal of Zhejiang Medical College as the carrier,Journal editorial department tried to improve students' scientific research ability.For instance,served fund projects,found competition's added value,offered course about research design and paper writing.The students' scientific research ability was improved.

Effects of Long-term Tai Ji Exercise on Risk Factors of Cardiovascular Diseases and Incidence of Chronic Disease / 中国康复理论与实践

@#Objective To explore the effects of long-term Tai Ji exercise on risk factors of cardiovascular diseases and incidence of chronic diseases. Methods The elderly involved were divided into control group (n=62) and Tai Ji group (n=63). Both of them received 2-years health education. The Tai Ji group exercised with the frequency of 30~40 minutes each time, 3 times a week, while the control group didn't change their daily behavior. They were observed 2 and 6 years later. Results 2 years later, the blood pressure, weight and waistline decreased in Tai Ji group compared with the control group (P<0.05). After 6 years followed, 1 people died and 4 people occured cardiovascular diseases in Tai Ji group, while 2 people died and 12 people occured cardiovascular diseases in the control group. The incidence of chronic diseases was lower in Tai Ji group (9.52%) than in the control group (33.87%) (P<0.01). And the blood pressure, waistline, and hipline in Tai Ji group decreased significantly compared with the control group (P<0.001). Conclusion Long-term Tai Ji Exercise can ameliorate the risk factors of cardiovascular diseases and reduce the incidence of chronic diseases.

Studies on macroscopic and microscopic identification of Cordyceps sinensis and its counterfeits / 中国中药杂志

<p><b>OBJECTIVE</b>To provide a rapid, simple, accurate and reproducible identification method from which Cordyceps sinensis can be distinguished from other species.</p><p><b>METHOD</b>To observe the larva and stroma of Cordyceps family with macroscopic identification method, and with powder microscopic identification method.</p><p><b>RESULT AND CONCLUSION</b>For macroscopic, only stroma of C. sinensis is mostly non-inflated, and un-obtuse at the tip, the caterpillar annulations of C. sinensis and the C. gracilis is distinct, about 20-30, and feet of above two are 8 pairs, 4 of 8 pairs are relatively distinct. The above appearance shows its unique characteristic. For microscopic identification, only C. sinensis exists microtrichia, the tip is pointed. The arranging order of stubby setae is irregular, the tip is blunt while the basal is gradually broader; the top of some setae bends slightly like a hook.</p>

Cytomegalovirus infects its host cells and induces their renin gene expression / 中华微生物学和免疫学杂志

Objective To explore renin expression in cytomegalovirus(CMV) infected juxtaglomerular cells(JG) and its biological significance. Methods JG model cell line As4.1 cells derived from kidney tissue were respectively incubated with murine CMV at multiplicities of infection(MOI) of 10, 0.1 and 0 for 5 d, and the control was mock infection with the same amount of ultraviolet inactive CMV as MOI 10, then the cells were harvested. CMV immediate early gene(IE1) mRNA in the cells was tested by RT-PCR. The renin positive cells and the renin fluorescence granules in the cells were examined by immunofluorescence stain. Whether or not re-nin antigen and CMV antigen were showed in the same cells by FITC and TRITC immunofluorescence. The renin gene expression in the cells was individually detected by real-time RT-PCR and Western blot. Results The cells infected by CMV showed typical cytopathic effect(CPE) and viral plaques in the cell monolayer. CMV IE1 mRNA was found in the viral infected cells by RT-PCR. The mass or ring granules of renin positive fluorescence appeared in the cytoplasm of the CPE cells. The renin positive cells congregated around the viral plaques. Renin positive granules and CMV positive granules showed in the same cells. Renin expression in the CMV infected cells exhibited in a dependent manner of ratio of infectious virus particles to cells. Conclusion CMV infection of the cells derived from kidney tissue induces renin expression related to a new pathogenesis of cardiovascular diseases.

Variation of hypervariable regions of the genome of hepatitis C virus in EBV transforming PBMC from patients with hepatitis C / 中华传染病杂志

Objective To study the persistence and replication of hepatitis C virus (HCV) RNA in Epstein Barr virus (EBV) transforming peripheral blood mononuclear cell (PBMC) from patients with he patitis C as well as variation of hypervariable regions (HVR) of the HCV genome. Methods PBMC from one patient with hepatitis C was infected by EBV and then transformed into lymphoblasts capable of being propagated indefinitely. Then, HCV RNA of the cultured cells and supernatants was detected by reverse transcriptase polymerase chain reaction (RT PCR) every month and in situ PCR to identify the location of HCV RNA in the cells. The HVR genome sequences of HCV in the first and the ninth month subculture cells were identified by sequence analysis. Results HCV plus strand RNA could be detected in the cultured cells for as long as 1 year. The HCV plus strand RNA could be identified in supernatants and the minus strand RNA were also observed in the cultured cells intermittently. In situ PCR showed that the blue black positive signals of HCV RNA located mainly in cytoplasmas of EBV transforming B cell but negative in nucleous. The positive signal was not found in negative control cells by in situ RT PCR. HCV HVR genome sequence after half year was found to have two regions of a high degree of variability in 1491 to 1583 nucleotide (384 to 414 amino acid) and 1761～1781 nucleotide (473 to 479 amino acid). But, compared the HCV HVR genome sequence in first month subcultured cells with that in ninth month, there was not significant difference. Conclusion HCV may exist in the cultured cell line for a prolonged period wihtout HVR genome sequence changed.