Cytosine Methylation Alteration in Natural Populations of Leymus chinensis Induced by Multiple Abiotic Stresses

Background

Human activity has a profound effect on the global environment and caused frequent occurrence of climatic fluctuations. To survive, plants need to adapt to the changing environmental conditions through altering their morphological and physiological traits. One known mechanism for phenotypic innovation to be achieved is environment-induced rapid yet inheritable epigenetic changes. Therefore, the use of molecular techniques to address the epigenetic mechanisms underpinning stress adaptation in plants is an important and challenging topic in biological research. In this study, we investigated the impact of warming, nitrogen (N) addition, and warming+nitrogen (N) addition stresses on the cytosine methylation status of Leymus chinensis Tzvel. at the population level by using the amplified fragment length polymorphism (AFLP), methylation-sensitive amplified polymorphism (MSAP) and retrotransposon based sequence-specific amplification polymorphism (SSAP) techniques.

Methodology/Principal Findings

Our results showed that, although the percentages of cytosine methylation changes in SSAP are significantly higher than those in MSAP, all the treatment groups showed similar alteration patterns of hypermethylation and hypomethylation. It meant that the abiotic stresses have induced the alterations in cytosine methylation patterns, and the levels of cytosine methylation changes around the transposable element are higher than the other genomic regions. In addition, the identification and analysis of differentially methylated loci (DML) indicated that the abiotic stresses have also caused targeted methylation changes at specific loci and these DML might have contributed to the capability of plants in adaptation to the abiotic stresses.

Conclusions/Significance

Our results demonstrated that abiotic stresses related to global warming and nitrogen deposition readily evoke alterations of cytosine methylation, and which may provide a molecular basis for rapid adaptation by the affected plant populations to the changed environments.     

Bienzymatic glucose biosensor based on co-immobilization of peroxidase and glucose oxidase on a carbon nanotubes electrode

A bienzymatic glucose biosensor was proposed for selective and sensitive detection of glucose. This mediatorless biosensor was made by simultaneous immobilization of glucose oxidase (GOD) and horseradish peroxidase (HRP) in an electropolymerized pyrrole (PPy) film on a single-wall carbon nanotubes (SWNT) coated electrode. The amperometric detection of glucose was assayed by potentiostating the bienzymatic electrode at -0.1 versus Ag/AgCl to reduce the enzymatically produced H(2)O(2) with minimal interference from the coexisting electroactive compounds. The single-wall carbon nanotubes, sandwiched between the enzyme loading polypyrrole (PPy) layer and the conducting substrate (gold electrode), could efficiently promote the direct electron transfer of HRP. Operational characteristics of the bienzymatic sensor, in terms of linear range, detection limit, sensitivity, selectivity and stability, were presented in detail.     

染色质调节元件与不同启动子的相互作用对基因表达调控的影响

为探究DNA序列元件对不同启动子调节转基因稳定表达的影响,利用遍在染色质开放元件 (Ubiquitous chromatin opening elements,UCOE) 和基质黏附序列 (Scaffold/matrix-attachment regions,MAR) 分别与含增强子的oct4基因启动子、含CpG岛的sox2基因启动子和不含调控元件的nanog基因启动子以及同时包含增强子和CpG岛的CMV启动子组合构建pOCT4-MAR、pOCT4-UCOE、pSOX2-MAR、pSOX2-UCOE、pNANOG-MAR、pNANOG-UCOE、pCMV-UCOE、pCMV-MAR等质粒,分析这些质粒稳定转染后的表达量和嵌合表达差异。结果发现,UCOE与含增强子元件的oct4启动子组合能较稳定高效表达,而MAR与含CpG岛的sox2启动子组合能较稳定高效表达。利用排除位置效应原因的嵌合表达对染色质高级结构调控基因表达的稳定性分析表明：(1) 通常情况下UCOE比MAR调节的表达载体的表达更高效和更稳定;UCOE连接含CpG岛的启动子形成开放染色质调节的高表达更稳定;(2) MAR与启动子上TATA盒或增强子可能通过染色质环产生高表达,但相对不稳定。结论：染色质调节元件UCOE和MAR与启动子调控元件之间能通过染色质开放状态或染色质环调控基因稳定表达。     

TIPE1‐mediated autophagy suppression promotes nasopharyngeal carcinoma cell proliferation via the AMPK/mTOR signalling pathway

Recent studies have shown that tumour necrosis factor‐α–induced protein 8 like‐1(TIPE1) plays distinct roles in different cancers. TIPE1 inhibits tumour proliferation and metastasis in a variety of tumours but acts as an oncogene in cervical cancer. The role of TIPE1 in nasopharyngeal carcinoma (NPC) remains unknown. Interestingly, TIPE1 expression was remarkably increased in NPC tissue samples compared to adjacent normal nasopharyngeal epithelial tissue samples in our study. TIPE1 expression was positively correlated with that of the proliferation marker Ki67 and negatively correlated with patient lifespan. In vitro, TIPE1 inhibited autophagy and induced cell proliferation in TIPE1‐overexpressing CNE‐1 and CNE‐2Z cells. In addition, knocking down TIPE1 expression promoted autophagy and decreased proliferation, whereas overexpressing TIPE1 increased the levels of pmTOR, pS6 and P62 and decreased the level of pAMPK and the LC3B. Furthermore, the decrease in autophagy was remarkably rescued in TIPE1‐overexpressing CNE‐1 and CNE‐2Z cells treated with the AMPK activator AICAR. In addition, TIPE1 promoted tumour growth in BALB/c nude mice. Taken together, results indicate that TIPE1 promotes NPC progression by inhibiting autophagy and inducing cell proliferation via the AMPK/mTOR signalling pathway. Thus, TIPE1 could potentially be used as a valuable diagnostic and prognostic biomarker for NPC.     

In Vitro Anti-hepatitis B Virus Activity of 2',3'-Dideoxyguanosine

2',3'-dideoxyguanosine(DoG) has been demonstrated to inhibit duck hepatitis B virus(DHBV) replication in vivo in a duck model of HBV infection. In the current study, the in vitro antiviral effects of DoG on human and animal hepadnaviruses were investigated. Our results showed that DoG effectively inhibited HBV, DHBV, and woodchuck hepatitis virus(WHV)replication in hepatocyte-derived cells in a dose-dependent manner, with 50% effective concentrations(EC50) of 0.3 ± 0.05, 6.82 ± 0.25, and 23.0 ± 1.5 lmol/L, respectively. Similar to other hepadnaviral DNA polymerase inhibitors,DoG did not alter the levels of intracellular viral RNA but induced the accumulation of a less-than-full-length viral RNA species, which was recently demonstrated to be generated by RNase H cleavage of pgRNA. Furthermore, using a transient transfection assay, DoG showed similar antiviral activity against HBV wild-type, 3TC-resistant rtA181 V, and adefovirresistant rtN236T mutants. Our results suggest that DoG has potential as a nucleoside analogue drug with anti-HBV activity.     

Deregulation of UCA1 expression may be involved in the development of chemoresistance to cisplatin in the treatment of non-small-cell lung cancer via regulating the signaling pathway of microRNA-495/NRF2

Non-small-cell lung cancer (NSCLC) remains the leading cause of cancer death worldwide. As a platinum-based chemotherapeutic drug, cisplatin has been used for over 30 years in NSCLC treatment while its effects are diminished by drug resistance. Therefore, we aimed to study the potential role of UCA1 in the development of chemoresistance against cisplatin. Real-time polymerase chain reaction, western-blot analysis, and immunofluorescence were used to study the involvement of UCA1, miR-495, and NRF2 in chemoresistance against cisplatin. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the effect of cisplatin on cell proliferation. Computational analysis and luciferase assay were carried out to explore the interaction among UCA1, miR-495, and NRF2. The cisplatin-R group exhibited lower levels of UCA1 and NRF2 expression but a higher level of miR-495 expression than the cisplatin-S group. The growth rate and half-maximal inhibitory concentration of cellular dipeptidyl peptidase (cisplatinum) of the cisplatin-R group were much higher than those in the cisplatin-S group. MiR-495 contained a complementary binding site of UCA1, and the luciferase activity of wild-type UCA1 was significantly reduced after the transfection of miR-495 mimics. MiR-495 directly targeted the 3′-untranslated region (3′-UTR) of NRF2, and the luciferase activity of wild-type NRF2 3′-UTR was evidently inhibited by miR-495 mimics. Finally, UCA1 and NRF2 expressions in the effective group were much lower than that in the ineffective group, along with a much higher level of miR-495 expression. We suggested for the first time that high expression of UCA1 contributed to the development of chemoresistance to cisplatin through the UCA1/miR-495/NRF2 signaling pathway.     

Immuno-spin trapping of protein and DNA radicals: “Tagging” free radicals to locate and understand the redox process

Biomolecule-centered radicals are intermediate species produced during both reversible (redox modulation) and irreversible (oxidative stress) oxidative modification of biomolecules. These oxidative processes must be studied in situ and in real time to understand the molecular mechanism of cell adaptation or death in response to changes in the extracellular environment. In this regard, we have developed and validated immuno-spin trapping to tag the redox process, tracing the oxidatively generated modification of biomolecules, in situ and in real time, by detecting protein- and DNA-centered radicals. The purpose of this methods article is to introduce and update the basic methods and applications of immuno-spin trapping for the study of redox biochemistry in oxidative stress and redox regulation. We describe in detail the production, detection, and location of protein and DNA radicals in biochemical systems, cells, and tissues, and in the whole animal as well, by using immuno-spin trapping with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide.     

Effects of emotional and physiological stress on plaque instability in apolipoprotein E knockout mice

In the present study, we sought to investigate the effects of emotional and physiological stress on plaque instability in atherosclerosis. We used different stress-treated apolipoprotein E (ApoE)-deficient mice, which have been shown to spontaneously develop atherosclerosis with features similar to those seen in humans, as an animal model. Morphology study showed that emotional stress (ES) obviously promoted the development of atherosclerotic plaques and plaque instability evidenced by significantly increasing plaque size, plaque-to-surface ratio and plaque calcification, and enhancing the frequency of large necrotic core and medial erosion compared with control ApoE^−/− mice (P < 0.01). Physiological stress (PS) treatment alone did not affect the plaque stability compared with control ApoE^−/− mice (P > 0.05). However, the combination of ES and PS treatment (CS) initiated much stronger plaque instability compared with ES treatment alone (P < 0.01), increased the frequency of thin fibrous caps, and even triggered plaque rupture and buried fibrous cap. Immunohistochemical analysis indicated that both ES and CS treatment led to an increase in the accumulation of macrophages and T cells and a decrease of smooth muscle cells, reflecting an unstable atherosclerotic plaque phenotype, in the atherosclerotic lesions in ApoE^−/− mice. PS alone did not affect plaque cellular components. Similarly, CS-mediated changes in atherosclerotic plaque composition were stronger than that caused by ES alone (P < 0.01). Taken together, ES treatment alone is sufficient to promote plaque instability. PS alone does not affect atherosclerotic plaque development, but can potentiate ES-mediated plaque destabilization.     

The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure

Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES) cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.