Changes in DNA methylation and transgenerational mobilization of a transposable element (mPing) by the topoisomerase II inhibitor, etoposide, in rice

ABSTRACT: BACKGROUND: Etoposide (epipodophyllotoxin) is a chemical commonly used as an anti-cancer drug which inhibits DNA synthesis by blocking topoisomerase II activity. Previous studies in animal cells have demonstrated that etoposide constitutes a genotoxic stress which may induce genomic instability including mobilization of normally quiescent transposable elements (TEs). However, it remained unknown whether similar genetically mutagenic effects could be imposed by etoposide in plant cells. Also, no information is available with regard to whether the drug may cause a perturbation of epigenetic stability in any organism. RESULTS: To investigate whether etoposide could generate genetic and/or epigenetic instability in plant cells, we applied etoposide to germinating seeds of six cultivated rice (Oryza sativa L.) genotypes including both subspecies, japonica and indica. Based on the methylation-sensitive gel-blotting results, epigenetic changes in DNA methylation of three TEs (Tos17, Osr23 and Osr36) and two protein-encoding genes (Homeobox and CDPK-related genes) were detected in the etoposide-treated plants (S0 generation) in four of the six studied japonica cultivars, Nipponbare, RZ1, RZ2, and RZ35, but not in the rest japonica cultivar (Matsumae) and the indica cultivar (93-11). DNA methylation changes in the etoposide-treated S0 rice plants were validated by bisulfite sequencing at both of two analyzed loci (Tos17 and Osr36). Transpositional activity was tested for eight TEs endogenous to the rice genome in both the S0 plants and their selfed progenies (S1 and S2) of one of the cultivars, RZ1, which manifested heritable phenotypic variations. Results indicated that no transposition occurred in the etoposide-treated S0 plants for any of the TEs. Nonetheless, a MITE transposon, mPing, showed rampant mobilization in the S1 and S2 progenies descended from the drug-treated S0 plants. CONCLUSIONS: Our results demonstrate that etoposide imposes a similar genotoxic stress on plant cells as it does on animal and human cells, which may induce transgenerational genomic instability by instigating transpositional activation of otherwise dormant TEs. In addition, we show for the first time that etoposide may induce epigenetic instability in the form of altered DNA methylation patterns in eukaryotes. However, penetration of the genotoxic effects of etoposide on plant cells, as being reflected as genetic and epigenetic instability, appears to be in a strictly genotype- and/or generation-dependent manner.     

Single nucleotide polymorphisms in Cryptomeria japonica: their discovery and validation for genome mapping and diversity studies

In order to develop a large set of single-nucleotide polymorphisms (SNPs) in Cryptomeria japonica, for a wide range of applications, we adopted a systematic EST (expressed sequence tags) re-sequencing approach. We examined a group of four genotypes comprising parents of a mapping population as well as representatives of two main lines from natural populations. We sequenced 5,170 gene fragments, representing analysis of over 1.3?Mb of DNA sequences in C. japonica. This analysis leads to the discovery of 13,413 SNPs in 3,744 amplicons, with an average of one SNP for every 101.0?bp (one SNP for every 78.3?bp in introns and for every 106.7?bp in exon regions). Nucleotide diversity in C. japonica (???=?0.0045) was found to be similar to values recorded in highly polymorphic forest tree species such as pine. We also validated the use of the SNPs as molecular markers for genetic diversity studies using the high throughput SNP genotyping platform GoldenGate. From 1,536 candidate SNP sites tested, 1,164 (75.8?%) were confirmed to be polymorphic. We anticipate that the genome-wide SNP markers reported here will be useful for evaluating the species?? range-wide genetic structure and in marker-assisted selection used as part of the C. japonica tree improvement program.     

Adult Mortality Attributable to Preventable Risk Factors for Non-Communicable Diseases and Injuries in Japan: A Comparative Risk Assessment

Background

The population of Japan has achieved the longest life expectancy in the world. To further improve population health, consistent and comparative evidence on mortality attributable to preventable risk factors is necessary for setting priorities for health policies and programs. Although several past studies have quantified the impact of individual risk factors in Japan, to our knowledge no study has assessed and compared the effects of multiple modifiable risk factors for non-communicable diseases and injuries using a standard framework. We estimated the effects of 16 risk factors on cause-specific deaths and life expectancy in Japan.

Methods and Findings

We obtained data on risk factor exposures from the National Health and Nutrition Survey and epidemiological studies, data on the number of cause-specific deaths from vital records adjusted for ill-defined codes, and data on relative risks from epidemiological studies and meta-analyses. We applied a comparative risk assessment framework to estimate effects of excess risks on deaths and life expectancy at age 40 y. In 2007, tobacco smoking and high blood pressure accounted for 129,000 deaths (95% CI: 115,000–154,000) and 104,000 deaths (95% CI: 86,000–119,000), respectively, followed by physical inactivity (52,000 deaths, 95% CI: 47,000–58,000), high blood glucose (34,000 deaths, 95% CI: 26,000–43,000), high dietary salt intake (34,000 deaths, 95% CI: 27,000–39,000), and alcohol use (31,000 deaths, 95% CI: 28,000–35,000). In recent decades, cancer mortality attributable to tobacco smoking has increased in the elderly, while stroke mortality attributable to high blood pressure has declined. Life expectancy at age 40 y in 2007 would have been extended by 1.4 y for both sexes (men, 95% CI: 1.3–1.6; women, 95% CI: 1.2–1.7) if exposures to multiple cardiovascular risk factors had been reduced to their optimal levels as determined by a theoretical-minimum-risk exposure distribution.

Conclusions

Tobacco smoking and high blood pressure are the two major risk factors for adult mortality from non-communicable diseases and injuries in Japan. There is a large potential population health gain if multiple risk factors are jointly controlled. Please see later in the article for the Editors'' Summary     

Systematic SAR study of the side chain of nonsecosteroidal vitamin D₃ analogs

A series of nonsecosteroidal vitamin D(3) analogs with carboxylic acid were explored. Through our systematic SAR studies on the side chain moiety, compound 6b was identified as the optimal compound showing excellent vitamin D receptor (VDR) agonistic activity. Compound 6b had the diethyl group in the terminal which was bound by (E)-olefin linker to the bisphenyl core. Calculating the volume of the side chain showed that the diethyl group in 6b filled the hydrophobic region of VDR with the ideal packing coefficient based on the 55% rule, and that this resulted in the most potent in vitro activity.     

Synthesis of β(1,3) oligoglucans exhibiting a Dectin-1 binding affinity and their biological evaluation

In this report, we describe the synthesis and biological evaluation of β(1,3) oligosaccharides that contain an aminoalkyl group and their biological evaluation. A 2,3 diol glycoside with a 4,6 benzylidene protecting group was used as an effective glycosyl acceptor for the synthesis of some β(1,3) linked glycosides. The use of a combination of a linear tetrasaccharide and a branched pentasaccharide as glycosyl donors led to the preparation of β(1,3) linear octa- to hexadecasaccharides and branched nona- to heptadecasaccharides in good total yields. Measurements of the competitive effects of the oligosaccharides on the binding of a soluble form of Dectin-1 to a solid-supported Schizophyllan (SPG) revealed that the branched heptadecasaccharide and the linear hexadecasaccharides also have binding activity for Dectin-1. In addition, the two oligosaccharides, both of which contain a β(1,3) hexadecasaccharide backbone, exhibited agonist activity in a luciferase-assisted NF-κB assay. STD-NMR analyses of complexes of Dectin-1 and the linear hexadecasaccharides clearly indicate Dectin-1 specifically recognizes the sugar part of the oligosaccharides and not the aminoalkyl chain.     

Construction of three quadruple-fluorescent MDA435 cell lines that enable monitoring of the whole chromosome segregation process in the living state

Mitotic events from prophase to telophase are defined by morphology or movement of chromatin, nuclear envelope, centrosomes and spindles. Live-cell imaging is useful for characterizing the whole chromosome segregation process in the living state. In this study, we constructed three quadruple-fluorescent MDA435 cell lines in which chromatin, kinetochores, nuclear envelope and either inner centromere, microtubules or centrosomes/spindles were differentially visualized with cyan, green, orange and red fluorescent proteins (ECFP, EGFP, mKO and DsRed). Each mitotic stage of the individual cells could be identified by capturing live-cell images without the requirement of fixing or staining steps. In addition, we obtained four-color time-lapse images of one cell line, MDA-Auro/imp/H3/AF, from prophase to metaphase and from early anaphase to telophase. These quadruple-fluorescent cell lines will be useful for precisely analyzing the mitotic events from prophase through to telophase in single cells in the future.     

Worker size in the formosan subterranean termite in relation to colony breeding structure as inferred from molecular markers

The Formosan subterranean termite, Coptotermes formosanus Shiraki, is an invasive species that originated in China and has been introduced to Hawaii and the U.S. mainland. Colonies are headed either by a pair of reproductives (simple families) or by varying numbers of inbreeding reproductives (extended families), and therefore have variable degrees of inbreeding. Worker size also varies among colonies of Formosan termites. We tested whether variation in worker size can be explained by the breeding system. Workers were collected from colonies from three geographically separated populations (China, Hawaii, and Louisiana), and body weight and head size were measured. Microsatellite genotyping was used to establish whether colonies were simple or extended families and to determine the heterozygosity of workers and their degree of inbreeding relative to their colony (F (IC), sensitive to the number of reproductives). All Chinese colonies contained multiple inbreeding neotenics. In Hawaii, 37% of the colonies were simple families and 63% were extended families, both having considerable degrees of inbreeding. In Louisiana, 57% of the colonies were simple families, which were mostly headed by unrelated pairs, and 43% were extended families. In simple families, size and body weight of workers were not associated with F (IC) or heterozygosity. In extended families of two populations, both size parameters were negatively correlated with F (IC); however, heterozygosity was not associated with worker size in any of the populations. This suggests that the number of reproductives within colonies has a stronger influence on worker size than the individuals' genetic diversity in Formosan subterranean termite colonies.