利用AFLP技术鉴定凤凰单丛古茶树种质资源

本文采用AFLP技术对34个凤凰单丛古茶树资源进行了种质鉴定分析.结果显示,筛选出多态性较高的5对引物中,引物组合E41M42、E41M39和E41M33可鉴别出供试的全部资源,鉴别效率为100%;有23份资源具有特异带,占供试材料的67.6%.由此研究表明,AFLP技术可以通过特异带、特异的谱带类型、不同引物提供谱带的组合将风凰单丛古茶树资源区分开来,这对保护凤凰单丛古茶树资源的育种产权、登录新品种、鉴定和检测其种子与苗木的真实性和纯度具有重要的现实意义.     

AFM‐ and NSOM‐based force spectroscopy and distribution analysis of CD69 molecules on human CD4+ T cell membrane

Although CD69 is well known as an early T cell‐activation marker, the possibility that CD69 are distributed as nano‐structures on membrane for immune regulation during T cell activation has not been tested. In this study, nanoscale features of CD69 expression on activated T cells were determined using the atomic force microscopy (AFM) topographic and force‐binding nanotechnology as well as near‐field scanning optical microscopy (NSOM)‐/fluorescence quantum dot (QD)‐based nanosacle imaging. Unstimulated CD4⁺ T cells showed neglectable numbers of membrane CD69 spots binding to the CD69 Ab‐functinalized AFM tip, and no detectable QD‐bound CD69 as examined by NSOM/QD‐based imaging. In contrast, Phytohemagglutinin (PHA)‐activated CD4⁺ T cells expressed CD69, and displayed many force‐binding spots binding to the CD69 Ab‐functionalized AFM tip on about 45% of cell membrane, with mean binding‐rupture forces 276 ± 71 pN. Most CD69 molecules appeared to be expressed as 100–200 nm nanoclusters on the membrane of PHA‐activated CD4⁺ T cells. Meanwhile, NSOM/QD‐based nanoscale imaging showed that CD69 were non‐uniformly distributed as 80–200 nm nanoclusters on cell‐membrane of PHA‐activated CD4⁺ T cells. This study represents the first demonstration of the nano‐biology of CD69 expression during T cell activation. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of noncytotoxic and IL-10-producing CD8+AT2R+ T cell population in response to ischemic heart injury

Emerging evidence suggests a cardioprotective role of the angiotensin AT2R, albeit the underlying cellular mechanisms are not well understood. We aimed in this article to elucidate a potential role of cardiac angiotensin AT2R in regulating cellular immune response to ischemic heart injury. Seven days after myocardial infarction in rats, double-immunofluorescence staining showed that AT2R was detected in a fraction of CD8(+) T cells infiltrating in the peri-infarct myocardium. We developed a method that allowed the isolation of myocardial infiltrating CD8(+)AT2R(+) T cells using modified MACS, and further characterization and purification with flow cytometry. Although the CD8(+)AT2R(-) T cells exhibited potent cytotoxicity to both adult and fetal cardiomyocytes (CMs), the CD8(+)AT2R(+) T cells were noncytotoxic to these CMs. The CD8(+)AT2R(+) T cells were characterized by upregulated IL-10 and downregulated IL-2 and INF-γ expression when compared with CD8(+)AT2R(-) T cells. We further showed that IL-10 gene expression was enhanced in CD8(+) T cells on in vitro AT2R stimulation. Importantly, in vivo AT2R activation engendered an increment of CD8(+)AT2R(+) T cells and IL-10 production in the ischemic myocardium. In addition, intramyocardial transplantation of CD8(+)AT2R(+) T cells (versus CD8(+)AT2R(-)) led to reduced ischemic heart injury. Moreover, the CD8(+)AT2R(+) T cell population was also demonstrated in human peripheral blood. Thus, we have defined the cardioprotective CD8(+)AT2R(+) T cell population, which increases during ischemic heart injury and contributes to maintaining CM viability and providing IL-10, hence revealing an AT2R-mediated cellular mechanism in modulating adaptive immune response in the heart.     

Multifunctional TENG for Blue Energy Scavenging and Self‐Powered Wind‐Speed Sensor

Triboelectric nanogenerator (TENG) has been considered to be a more effective technology to harvest various types of mechanic vibration energies such as wind energy, water energy in the blue energy, and so on. Considering the vast energy from the blue oceans, harvesting of the water energy has attracted huge attention. There are two major types of “mechanical” water energy, water wave energy in random direction and water flow kinetic energy. However, although the most reported TENG can be used to efficiently harvest one type of water energy, to simultaneously collect two or more types of such energy still remains challenging. In this work, two different freestanding, multifunctional TENGs are successfully developed that can be used to harvest three types of energies including water waves, air flowing, and water flowing. These two new TENGs designed in accordance with the same freestanding model yield the output voltages of 490 and ≈100 V with short circuit currents of 24 and 2.7 µA, respectively, when operated at a rotation frequency of 200 rpm and the movement frequency of 3 Hz. Moreover, the developed multifunctional TENG can also be explored as a self‐powered speed sensor of wind by correlating the short‐circuit current with the wind speed.     

絮凝活性产紫青霉EL-02的分离鉴定及其絮凝活性初探

【目的】分离鉴定有絮凝活性真菌,同时对其絮凝活性进行初步研究。【方法】采用梯度稀释、平板划线、18SrDNA检测等方法分离鉴定絮凝活性菌株。通过高速离心、超声破碎、乙醇沉淀、定性试验等方法确定絮凝活性物质性质。【结果】从渤海湾海岸土壤样品中分离筛选出一株有较高絮凝活性的真菌,经鉴定为产紫青霉(Penicillium purpurogenum),命名为产紫青霉EL-02(P.purpurogenum EL-02)。超声破碎试验证实其絮凝活性主要存在于发酵上清液。根据絮凝活性曲线,确定4d为积累絮凝活性产物的最佳发酵时间。乙醇沉淀法获得该菌株絮凝活性物质。经鉴定该菌株所产絮凝活性物质为糖类,且其活性在pH2-11,温度-70℃-100℃范围内保持稳定。【结论】分离筛选到一株有絮凝活性的产紫青霉EL-02,经鉴定其产生糖类絮凝活性物质。     

Active tolerance induction and prevention of autoimmune diabetes by immunogene therapy using recombinant adenoassociated virus expressing glutamic acid decarboxylase 65 peptide GAD(500-585)

Tolerance induction of autoreactive T cells against pancreatic beta cell-specific autoantigens such as glutamic acid decarboxylase 65 (GAD65) and insulin has been attempted as a method to prevent autoimmune diabetes. In this study, we investigate whether adenoassociated virus (AAV) gene delivery of multiple immunodominant epitopes expressing GAD(500-585) could induce potent immune tolerance and persistently suppress autoimmune diabetes in NOD mice. A single muscle injection of 7-wk-old female NOD mice with rAAV/GAD(500-585) (3 x 10(11) IU/mouse) quantitatively reduced pancreatic insulitis and efficiently prevented the development of overt type I diabetes. This prevention was marked by the inactivation of GAD(500-585)-responsive T lymphocytes, the enhanced GAD(500-585)-specific Th2 response (characterized by increased IL-4, IL-10 production, and decreased IFN-gamma production; especially elevated anti-GAD(500-585) IgG1 titer; and relatively unchanged anti-GAD(500-585) IgG2b titer), the increased secretion of TGF-beta, and the production of protective regulatory cells. Our studies also revealed that peptides 509-528, 570-585, and 554-546 in the region of GAD(500-585) played important roles in rAAV/GAD(500-585) immunization-induced immune tolerance. These data indicate that using AAV, a vector with advantage for therapeutic gene delivery, to transfer autoantigen peptide GAD(500-585), can induce immunological tolerance through active suppression of effector T cells and prevent type I diabetes in NOD mice.     

White Pitaya (Hylocereus undatus) Juice Attenuates Insulin Resistance and Hepatic Steatosis in Diet-Induced Obese Mice

Insulin resistance and hepatic steatosis are the most common complications of obesity. Pitaya is an important source of phytochemicals such as polyphenols, flavonoid and vitamin C which are related to its antioxidant activity. The present study was conducted to evaluate the influence of white pitaya juice (WPJ) on obesity-related metabolic disorders (e.g. insulin resistance and hepatic steatosis) in high-fat diet-fed mice. Forty-eight male C57BL/6J mice were assigned into four groups and fed low-fat diet with free access to water or WPJ, or fed high-fat diet with free access to water or WPJ for 14 weeks. Our results showed that administration of WPJ improved high-fat diet-induced insulin resistance, hepatic steatosis and adipose hypertrophy, but it exerted no influence on body weight gain in mice. Hepatic gene expression analysis indicated that WPJ supplement not only changed the expression profile of genes involved in lipid and cholesterol metabolism (Srebp1, HMGCoR, Cpt1b, HL, Insig1 and Insig2) but also significantly increased the expression levels of FGF21-related genes (Klb, FGFR2, Egr1 and cFos). In conclusion, WPJ protected from diet-induced hepatic steatosis and insulin resistance, which was associated with the improved FGF21 resistance and lipid metabolism.     

High-quality Fagopyrum esculentum genome provides insights into the flavonoid accumulation among different tissues and self-incompatibility

Common buckwheat (Fagopyrum esculentum) and Tartary buckwheat (Fagopyrum tataricum), the two most widely cultivated buckwheat species, differ greatly in flavonoid content and reproductive mode. Here, we report the first high-quality and chromosome-level genome assembly of common buckwheat with 1.2 Gb. Comparative genomic analysis revealed that common buckwheat underwent a burst of long terminal repeat retrotransposons insertion accompanied by numerous large chromosome rearrangements after divergence from Tartary buckwheat. Moreover, multiple gene families involved in stress tolerance and flavonoid biosynthesis such as multidrug and toxic compound extrusion (MATE) and chalcone synthase (CHS) underwent significant expansion in buckwheat, especially in common buckwheat. Integrated multi-omics analysis identified high expression of catechin biosynthesis-related genes in flower and seed in common buckwheat and high expression of rutin biosynthesis-related genes in seed in Tartary buckwheat as being important for the differences in flavonoid type and content between these buckwheat species. We also identified a candidate key rutin-degrading enzyme gene (Ft8.2377) that was highly expressed in Tartary buckwheat seed. In addition, we identified a haplotype-resolved candidate locus containing many genes reportedly associated with the development of flower and pollen, which was potentially related to self-incompatibility in common buckwheat. Our study provides important resources facilitating future functional genomics-related research of flavonoid biosynthesis and self-incompatibility in buckwheat.     

Overcoming resistance to mitochondrial apoptosis by BZML‐induced mitotic catastrophe is enhanced by inhibition of autophagy in A549/Taxol cells

Objectives

Our previous in vitro study showed that 5‐(3, 4, 5‐trimethoxybenzoyl)‐4‐methyl‐2‐(p‐tolyl) imidazol (BZML) is a novel colchicine binding site inhibitor with potent anti‐cancer activity against apoptosis resistance in A549/Taxol cells through mitotic catastrophe (MC). However, the mechanisms underlying apoptosis resistance in A549/Taxol cells remain unknown. To clarify these mechanisms, in the present study, we investigated the molecular mechanisms of apoptosis and autophagy, which are closely associated with MC in BZML‐treated A549 and A549/Taxol cells.

Methods

Xenograft NSCLC models induced by A549 and A549/Taxol cells were used to evaluate the efficacy of BZML in vivo. The activation of the mitochondrial apoptotic pathway was assessed using JC‐1 staining, Annexin V‐FITC/PI double‐staining, a caspase‐9 fluorescence metric assay kit and western blot. The different functional forms of autophagy were distinguished by determining the impact of autophagy inhibition on drug sensitivity.

Results

Our data showed that BZML also exhibited desirable anti‐cancer activity against drug‐resistant NSCLC in vivo. Moreover, BZML caused ROS generation and MMP loss followed by the release of cytochrome c from mitochondria to cytosol in both A549 and A549/Taxol cells. However, the ROS‐mediated apoptotic pathway involving the mitochondria that is induced by BZML was only fully activated in A549 cells but not in A549/Taxol cells. Importantly, we found that autophagy acted as a non‐protective type of autophagy during BZML‐induced apoptosis in A549 cells, whereas it acted as a type of cytoprotective autophagy against BZML‐induced MC in A549/Taxol cells.

Conclusions

Our data suggest that the anti‐apoptosis property of A549/Taxol cells originates from a defect in activation of the mitochondrial apoptotic pathway, and autophagy inhibitors can potentiate BZML‐induced MC to overcome resistance to mitochondrial apoptosis.