利用Inverse PCR快速筛选单个T-DNA拷贝转基因水稻植株的方法

为了获得单个T-DNA插入拷贝的植株, 我们建立了一套利用Inverse PCR(IPCR)快速检测转基因水稻中T-DNA拷贝数的方法。用IPCR的方法可以扩增出与已知T-DNA序列相邻的水稻基因组DNA未知序列,由此推测转基因水稻植株中T-DNA的拷贝数。我们共对15个转化株系20棵不同植株的DNA进行了IPCR检测。其中12株表现为T-DNA单拷贝插入,3株为双拷贝插入,1株为三拷贝插入。另外4株未检测到T-DNA插入拷贝。IPCR分析结果经过Southern杂交和测序的验证。     

Effects of Intra-Operative Total Intravenous Anaesthesia with Propofol versus Inhalational Anaesthesia with Sevoflurane on Post-Operative Pain in Liver Surgery: A Retrospective Case-Control Study

Background

Patients receiving total intravenous anesthesia (TIVA) with propofol have been shown to experience less postoperative pain. We evaluated the post-operative analgesic effects of propofol compared with sevoflurane maintenance of anesthesia in liver surgery. This study was registered at ClinicalTrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT02179437","term_id":"NCT02179437"}}NCT02179437).

Methods

In this retrospective study, records of patients who underwent liver surgery between 2010 and 2013 were reviewed. Ninety-five patients anesthetized with propofol TIVA were matched with 95 patients anesthetized with sevoflurane. Numeric pain rating scale (NRS) pain scores, postoperative morphine consumption, side effects and patients’ satisfaction with pain relief were evaluated.

Results

The TIVA group reported lower NRS pain scores during coughing on postoperative days 1 and 2 but not 3 (p = 0.0127, p = 0.0472, p = 0.4556 respectively). They also consumed significantly less daily (p = 0.001 on day 1, p = 0.0231 on day 2, p = 0.0004 on day 3), accumulative (p = 0.001 on day 1, p<0.0001 on day 2 and p = 0.0064 on day 3) and total morphine (p = 0.03) when compared with the sevoflurane group. There were no differences in total duration of intravenous patient controlled analgesia (PCA) morphine use and patient satisfaction. No difference was found in reported side effects.

Conclusion

Patients anesthetized with propofol TIVA reported less pain during coughing and consumed less daily, accumulative and total morphine after liver surgery.     

Host‐induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat

Fusarium head blight (FHB) and Fusarium seedling blight (FSB) of wheat, caused by Fusarium pathogens, are devastating diseases worldwide. We report the expression of RNA interference (RNAi) sequences derived from an essential Fusarium graminearum (Fg) virulence gene, chitin synthase (Chs) 3b, as a method to enhance resistance of wheat plants to fungal pathogens. Deletion of Chs3b was lethal to Fg; disruption of the other Chs gene family members generated knockout mutants with diverse impacts on Fg. Comparative expression analyses revealed that among the Chs gene family members, Chs3b had the highest expression levels during Fg colonization of wheat. Three hairpin RNAi constructs corresponding to the different regions of Chs3b were found to silence Chs3b in transgenic Fg strains. Co‐expression of these three RNAi constructs in two independent elite wheat cultivar transgenic lines conferred high levels of stable, consistent resistance (combined type I and II resistance) to both FHB and FSB throughout the T₃ to T₅ generations. Confocal microscopy revealed profoundly restricted mycelia in Fg‐infected transgenic wheat plants. Presence of the three specific short interfering RNAs in transgenic wheat plants was confirmed by Northern blotting, and these RNAs efficiently down‐regulated Chs3b in the colonizing Fusarium pathogens on wheat seedlings and spikes. Our results demonstrate that host‐induced gene silencing of an essential fungal chitin synthase gene is an effective strategy for enhancing resistance in crop plants under field test conditions.     

Determining an Optimal Cutoff of Serum β-Human Chorionic Gonadotropin for Assisting the Diagnosis of Intracranial Germinomas

Background

Beta (β)-human chorionic gonadotropin (β-HCG) is used to confirm the diagnosis and plan treatment of intracranial germinomas. However, the cutoff values of serum β-HCG in diagnosis of intracranial germinomas reported in the literature are inconsistent. To establish an appropriate cutoff value of serum β-HCG for diagnosis of intracranial germinomas, we retrospectively reviewed the records of intracranial tumor patients who received serum β-HCG and α-fetoprotein (AFP) tests for diagnostic purposes at our hospital from 2005 to 2014.

Methods

A total of 93 intracranial germinomas and 289 intracranial non-germ cell tumors were included in this study. Receiver operating characteristic (ROC) analysis was used to evaluate the sensitivity and specificity of 3 cutoffs (0.1, 0.4, and 0.5 mIU/mL) for diagnosing intracranial germinomas. The serum β-HCG level of intracranial germinoma patients was further analyzed to investigate the effect of metastasis status and tumor location on serum β-HCG level.

Results

The area under the ROC curve was 0.81 (P < .001), suggesting β-HCG is an effective marker. Of the 3 cutoff values, 0.1 mIU/mL possessed a highest sensitivity (66.67%) and good specificity (91%). Although there was no β-HCG level difference between metastatic and non-metastatic intracranial germinoma patients, the diagnostic rate of metastatic neurohypophyseal germinomas was significantly higher than that of its non-metastatic counterpart (P < .05), implying that the location of the germinoma might need to be considered when β-HCG is used as a marker to predict metastasis.

Conclusions

Determining an optimal cutoff of serum β-HCG is helpful for assisting the diagnosis of intracranial germinoma.     

大果木姜子的甘油三酯组成

采用光谱法和胰酯酶分解法对大果木姜子油酯中分得的结晶C—I组成进行了分析,结果表明C—I是由11种甘油三酯组成。其中2-位为月硅酸的甘油三酯占91.80％,1,3-位脂肪酸主要为癸酸和月桂酸,甘油三酯的组成中,CLC占62.24％,LLC占24.50％。     

Regulation of cell proliferation by fast Myosin light chain 1 in myoblasts derived from extraocular muscle, diaphragm and gastrocnemius

The extraocular muscle (EOM) suffers much less injury from Duchenne muscular dystrophy (DMD) than other skeletal muscles such as diaphragm and gastrocnemius. The present study was undertaken to test the hypothesis that differential expression of regulatory proteins between the EOM and other skeletal muscles is responsible for the observed difference in the sensitivity to DMD-associated damage. Protein expression in the tissue samples obtained from EOM, diaphragm or gastrocnemius of C57BL/6 mice was analyzed by two-dimensional gel electrophoresis and mass spectrometry. There were 35 proteins that were identified to be differentially expressed among different skeletal muscle tissues. Among the 35 proteins, a fast skeletal muscle isoform myosin light chain 1 (MLC1f) protein was further studied in relation to muscle cell proliferation. The EOM-derived myoblasts had much lower levels of MLC1f and higher rate of cell proliferation in contrast to the myoblasts derived from diaphragm or gastrocnemius, which displayed a higher expression of MLC1f along with a slow proliferation. Deletion of MLC1f using siRNA targeting MLC1f resulted in an increased rate of cell proliferation in the myoblasts. Cell cycle analysis revealed that MLC1f inhibited the transition of the cell cycle from the G1 to the S phase. Therefore, the present study demonstrates that MLC1f may negatively regulate proliferation of myoblasts through inhibition of the transition from the G1 to the S phase of the cell cycle. Low levels of MLC1f in myoblasts of EOM may ensure cell proliferation and enhance the repair process for EOM under the DMD disease condition, thus making EOM suffer less injury from DMD.     

Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis

Very little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis-associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis-related function of candidate protein interleukin-18 (IL-18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach was used to compare the protein expression profiles between PLA801C and PLA801D sublines. Eleven proteins were identified and further verified by one-dimensional Western blotting, Northern blot and/or semiquantitative reverse transciptase polymerase chain reaction analysis. Compared with those in poorly metastatic PLA801C subline, cytokeratin 18, tissue transglutaminase, Rho GDP-dissociation inhibitor 1, tropomyosin, fibroblast type, IL-18 and annexin I were significantly up-regulated, while protein disulfide isomerase, heat shock protein 60, peroxiredoxin 1, chlorine intracellular channel protein 1 (CLI1) and creatine kinase, B chain were significantly down-regulated in the highly metastatic PLA801D subline. Intriguingly, all the identified candidate proteins except for CLI1 have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was present in highly metastatic PLA801D but absent in poorly metastatic PLA801C, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense/IL-18 antisense into PLA801C/PLA801D sublines simultaneously. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro and down-regulated E-cadherin expression of PLA801C transfectants, while IL-18 antisense remarkably decreased cell invasion potency in vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play a role in metastasis by inhibiting E-cadherin expression.     

Distinct functions of two FabA-like dehydratase domains of polyunsaturated fatty acid synthase in the biosynthesis of very long-chain polyunsaturated fatty acids

Thraustochytrium is a unicellular marine protist for the commercial production of very long-chain polyunsaturated fatty acids (VLCPUFAs). Biosynthesis of these VLCPUFAs in the protist is catalysed by a PUFA synthase comprising three subunits, each with multiple catalytic domains. Among these domains, two tandem FabA-like dehydratase domains (DH1 and DH2) in subunit-C together are responsible for introducing double bonds in VLCPUFAs. Domain swapping analysis in yeast showed that the defective phenotype of a Scfas1 mutant could be complemented by expressing an engineered ScFAS1 gene in which the DH domain was replaced by a single DH1 or mutated DH2 of the two. Heterologous expression of the PUFA synthase in E. coli showed that the mutation of DH1 of the two or deletion of DH1 or substitution of DH1 with DH2 resulted in the complete loss of activity in the biosynthesis of VLCPUFAs. Mutation of DH2 of the two or deletion of the DH2 domain produced a small amount of DPA, but not docosahexaenoic acid (DHA). These results indicate that each of the two FabA-like domains of the PUFA synthase possesses distinct function. DH1 domain is essential for the biosynthesis of VLCPUFAs, but DH2 domain is required for the biosynthesis of DHA.     

黏附分子在肿瘤发生及发展中的作用

细胞黏附分子是以配体和受体相结合的形式,介导细胞与细胞间或细胞与基质间相互作用的一类分子,参与机体的多种重要生理和病理过程.近年来,在对肿瘤发生和发展的研究中发现,黏附分子可通过多种途径影响肿瘤的生长、浸润及转移过程.因此.对黏附分子在肿瘤发生和发展中作用及机制的深入研究,可为肿瘤早期诊断提供重要的分子指标和发现新的治疗靶标.并为进而形成临床诊疗新策略提供重要理论支持.现就几种重要黏附分子在肿瘤生长与转移中的作用进行综述.