豹蛙核酸酶与斑蝥酸钠对肺腺癌细胞增殖的协同抑制作用

豹蛙核酸酶(onconase,Onc)是从美洲北方豹蛙卵母细胞中提取的一种核糖核酸酶,对许多肿瘤细胞都具有杀伤作用。斑蝥素(cantharidin)是存在于芫青科昆虫斑蝥体内的一种天然防御性毒素,斑蝥酸钠(sodium cantharidate,SCA)是斑蝥素半合成衍生物。鉴于Onc与SCA对非小细胞肺癌都具有杀伤作用,采用MTT法测定Onc与SCA单独与联合作用于两株肺腺癌细胞的IC50值,运用联合作用指数(combination index,CI)和等效线分析评价两者联合作用的效果。结果表明,Onc与SCA联合作用时,CI值均小于0.7,等效线分析图显示,代表Onc与SCA联合作用的点均位于加成线下方,Onc与SCA对肺腺癌SPC-A-1、A549细胞株增殖的抑制作用具有协同效应。用流式细胞仪进行的凋亡细胞检测结果也支持上述"Onc/SCA联合使用具有协同抗癌作用"的结论。     

Comparative study of the cytotoxicity of the nanosized and microsized tellurium powders on HeLa cells

To compare the cytotoxicity on HeLa cells induced by nanosized and microsized tellurium powders, HeLa cells were exposed to different concentrations of tellurium powders (0, 50, 100, 150 and 200 μg/mL) for 12 h. In this study, detection of a series of biomarkers, including reactive oxygen species (ROS), glutathione (GSH), 8-hydroxy-2′-deoxyguanosine (8-OHdG), in addition to DNA and protein crosslink (DPC) and MTTassay, were conducted to evaluate the cytotoxicity. It is indicated that compared with the control group, there was no significant difference in the induced cytotoxicity at concentrations lower than 50 μg/mL for both nanosized and microsized tellurium powders. While there appears a significant difference in the induced cytotoxicity for nanosized tellurium powders when the concentration is higher than 100 μg/mL as well as for microsized tellurium powders when the concentration is higher than 200 μg/mL. Moreover, it is found that the cytotoxicity induced on HeLa cells exhibits a certain dose-effect relationship with the concentration of tellurium powders. A conclusion has been reached that the toxicity on HeLa cells can be induced by both nanosized and microsized tellurium powders, and the toxicity of the nanosized tellurium powders is significantly greater than the microsized one.     

单细胞技术在干细胞研究中的应用

干细胞是具有自我更新和分化潜能的异质性细胞群体。基于细胞群体水平的干细胞研究不能满足深入认识干细胞生物学本质及实际应用的需要。近年来,单细胞相关技术不断发展和成熟,并正在干细胞基础研究及其相关领域中获得迅速应用。该文以造血干细胞为主要例举,就实验研究中常用的单细胞分离、单细胞克隆分析、单细胞移植、单细胞实时定量PCR及单细胞测序等技术原理及其应用进行综述。     

Insecticidal activity of the chitinase from the Spodoptera litura nucleopolyhedrovirus

Baculovirus chitinase gene (chiA) is a late gene essential for liquefying the host insect at a late stage of infection for its hydrolyzing chitin function. In a previous report, baculovirus ChiA has been shown to offer many interesting new opportunities for pest control. Recently, a putative chiA gene was identified in the Korean isolate of the Spodoptera litura nucleopolyhedorvirus (SpliMNPV‐K1) genome. The open reading frame (ORF) contains 1692 nucelotides and encodes a protein of 563 amino acids with a predicted molecular weight of about 62.6 kDa. To study the insecticidal activity of ChiA from SpliMNPV‐K1, we constructed a recombinant AcMNPV, Ap‐SlChiA, which is designed to express the ChiA under the control of a polyhedrin promoter. Western blot analysis indicated that ChiA was successfully expressed by this recombinant virus. Chitinase assay revealed that the chitobiosidase and endochitinase activity of the recombinant virus was 2.5‐ and 3.9‐flods higher than those of wild‐type AcMNPV, respectively. In addition, the recombinant virus showed higher evident insecticidal activity against 3rd instar larvae of Spodotera exigua than that of the AcMNPV. These results suggest that the chiA gene from SpliMNPV‐K1 could be successfully applied to improve pathogenicity of baculoviruses.     

The stacked over-expression of FPS, CYP71AV1 and CPR genes leads to the increase of artemisinin level in Artemisia annua L.

Artemisinin is an endoperoxide sesquiterpene lactone isolated from the aerial parts of Artemisia annua L., and is presently the most potent anti-malarial drug. Owing to the low yield of artemisinin from A. annua as well as the widespread application of artemisinin-based combination therapy recommended by the World Health Organization, the global demand for artemisinin is substantially increasing and is therefore rendering artemisinin in short supply. An economical way to increase artemisinin production is to increase the content of artemisinin in A. annua. In this study, three key genes in the artemisinin biosynthesis pathway, encoding farnesyl diphosphate synthase, amorpha-4, 11-diene C-12 oxidase and its redox partner cytochrome P450 reductase, were over-expressed in A. annua through Agrobacterium-mediated transformation. The transgenic lines were confirmed by Southern blotting and the over-expressions of the genes were demonstrated by real-time PCR assays. The HPLC analysis showed that the artemisinin contents in transgenic lines were increased significantly, with the highest one found to be 3.6-fold higher (2.9 mg/g FW) than that of the control. These results demonstrate that multigene engineering is an effective way to enhance artemisinin content in A. annua.     

Expression of the Arabidopsis vacuolar H+-pyrophosphatase gene AVP1 in peanut to improve drought and salt tolerance

The Arabidopsis gene AVP1 encodes an H⁺-pyrophosphatase that functions as a proton pump at the vacuolar membranes, generating a proton gradient across vacuolar membranes, which serves as the driving force for many secondary transporters on vacuolar membranes such as Na⁺/H⁺-antiporters. Overexpression of AVP1 could improve drought tolerance and salt tolerance in transgenic plants, suggesting a possible way in improving drought and salt tolerance in crops. The AVP1 was therefore introduced into peanut by Agrobacterium-mediated transformation. Analysis of AVP1-expressing peanut indicated that AVP1-overexpression in peanut could improve both drought and salt tolerance in greenhouse and growth chamber conditions, as AVP1-overexpressing peanuts produced more biomass and maintained higher photosynthetic rates under both drought and salt conditions. In the field, AVP1-overexpressing peanuts also outperformed wild-type plants by having higher photosynthetic rates and producing higher yields under low irrigation conditions.     

Effects of AgRP Inhibition on Energy Balance and Metabolism in Rodent Models

Activation of brain melanocortin-4 receptors (MC4-R) by α-melanocyte-stimulating hormone (MSH) or inhibition by agouti-related protein (AgRP) regulates food intake and energy expenditure and can modulate neuroendocrine responses to changes in energy balance. To examine the effects of AgRP inhibition on energy balance, a small molecule, non-peptide compound, TTP2515, developed by TransTech Pharma, Inc., was studied in vitro and in rodent models in vivo. TTP2515 prevented AgRP from antagonizing α-MSH-induced increases in cAMP in HEK 293 cells overexpressing the human MC4-R. When administered to rats by oral gavage TTP2515 blocked icv AgRP-induced increases in food intake, weight gain and adiposity and suppression of T4 levels. In both diet-induced obese (DIO) and leptin-deficient mice, TTP2515 decreased food intake, weight gain, adiposity and respiratory quotient. TTP2515 potently suppressed food intake and weight gain in lean mice immediately after initiation of a high fat diet (HFD) but had no effect on these parameters in lean chow-fed mice. However, when tested in AgRP KO mice, TTP2515 also suppressed food intake and weight gain during HFD feeding. In several studies TTP2515 increased T4 but not T3 levels, however this was also observed in AgRP KO mice. TTP2515 also attenuated refeeding and weight gain after fasting, an effect not evident in AgRP KO mice when administered at moderate doses. This study shows that TTP2515 exerts many effects consistent with AgRP inhibition however experiments in AgRP KO mice indicate some off-target effects of this drug. TTP2515 was particularly effective during fasting and in mice with leptin deficiency, conditions in which AgRP is elevated, as well as during acute and chronic HFD feeding. Thus the usefulness of this drug in treating obesity deserves further exploration, to define the AgRP dependent and independent mechanisms by which TTP2515 exerts its effects on energy balance.     

Effect of Tenofovir on Nucleotidases and Cytokines in HIV-1 Target Cells

Tenofovir (TFV) has been widely used for pre-exposure prophylaxis of HIV-1 infection with mixed results. While the use of TFV in uninfected individuals for prevention of HIV-1 acquisition is actively being investigated, the possible consequences of TFV exposure for the HIV-target cells and the mucosal microenvironment are unknown. In the current study, we evaluated the effects of TFV treatment on blood-derived CD4⁺ T cells, monocyte-derived macrophages and dendritic cells (DC). Purified HIV-target cells were treated with different concentrations of TFV (0.001-1.0 mg/ml) for 2 to 24hr. RNA was isolated and RT-PCR was performed to compare the levels of mRNA expression of nucleotidases and pro-inflammatory cytokine genes (MIP3α, IL-8 and TNFα) in the presence or absence of TFV. We found that TFV increases 5’-ecto-nucleotidase (NT5E) and inhibits mitochondrial nucleotidase (NT5M) gene expression and increases 5’ nucleotidase activity in macrophages. We also observed that TFV stimulates the expression and secretion of IL-8 by macrophages, DC, and activated CD4⁺ T cells and increases the expression and secretion of MIP3α by macrophages. In contrast, TFV had no effect on TNFα secretion from macrophages, DC and CD4⁺ T cells. Our results demonstrate that TFV alters innate immune responses in HIV-target cells with potential implications for increased inflammation at mucosal surfaces. As new preventive trials are designed, these findings should provide a foundation for understanding the effects of TFV on HIV-target cells in microbicide trials.     

An Integrated Approach to Uncover Driver Genes in Breast Cancer Methylation Genomes

Background

Cancer cells typically exhibit large-scale aberrant methylation of gene promoters. Some of the genes with promoter methylation alterations play “driver” roles in tumorigenesis, whereas others are only “passengers”.

Results

Based on the assumption that promoter methylation alteration of a driver gene may lead to expression alternation of a set of genes associated with cancer pathways, we developed a computational framework for integrating promoter methylation and gene expression data to identify driver methylation aberrations of cancer. Applying this approach to breast cancer data, we identified many novel cancer driver genes and found that some of the identified driver genes were subtype-specific for basal-like, luminal-A and HER2+ subtypes of breast cancer.

Conclusion

The proposed framework proved effective in identifying cancer driver genes from genome-wide gene methylation and expression data of cancer. These results may provide new molecular targets for potential targeted and selective epigenetic therapy.