In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein

<p>Abstract</p> <p>Background</p> <p>Cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. An increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. Although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. Here, we developed <it>in planta </it>assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcriptionally silenced by an epigenetic mechanism.</p> <p>Results</p> <p>Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (<it>CHS-A</it>) gene and contains <it>CHS-A </it>transgenes whose transcription is repressed; <it>Nicotiana benthamiana </it>plants that contain the green fluorescent protein (<it>GFP</it>) reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in <it>Arabidopsis </it>plants.</p> <p>Conclusions</p> <p>Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes.</p

r逐次更新型作業者モデルによる行動予測とその不確実性に基づく協働ロボット向け動作計画

Tohoku University小菅一弘課

Synergistic effect of surface phosphorylation and micro-roughness on enhanced osseointegration ability of poly(ether ether ketone) in the rabbit tibia

This study was aimed to investigate the osseointegration ability of poly(ether ether ketone) (PEEK) implants with modified surface roughness and/or surface chemistry. The roughened surface was prepared by a sandblast method, and the phosphate groups on the substrates were modified by a two-step chemical reaction. The in vitro osteogenic activity of rat mesenchymal stem cells (MSCs) on the developed substrates was assessed by measuring cell proliferation, alkaline phosphatase activity, osteocalcin expression, and bone-like nodule formation. Surface roughening alone did not improve MSC responses. However, phosphorylation of smooth substrates increased cell responses, which were further elevated in combination with surface roughening. Moreover, in a rabbit tibia implantation model, this combined surface modification significantly enhanced the bone-to-implant contact ratio and corresponding bone-to-implant bonding strength at 4 and 8 weeks post-implantation, whereas modification of surface roughness or surface chemistry alone did not. This study demonstrates that combination of surface roughness and chemical modification on PEEK significantly promotes cell responses and osseointegration ability in a synergistic manner both in vitro and in vivo. Therefore, this is a simple and promising technique for improving the poor osseointegration ability of PEEK-based orthopedic/dental implants

DV200 Index for Assessing RNA Integrity in Next-Generation Sequencing

Poor quality of biological samples will result in an inaccurate analysis of next-generation sequencing (NGS). Therefore, methods to accurately evaluate sample integrity are needed. Among methods for evaluating RNA quality, the RNA integrity number equivalent (RINe) is widely used, whereas the DV200, which evaluates the percentage of fragments of >200 nucleotides, is also used as a quality assessment standard. In this study, we compared the RINe and DV200 RNA quality indexes to determine the most suitable RNA index for the NGS analysis. Seventy-one RNA samples were extracted from formalin-fixed paraffin-embedded tissue samples (n=30), fresh-frozen samples (n=25), or cell lines (n=16). After assessing RNA quality using the RINe and DV200, we prepared two kinds of stranded mRNA sequencing libraries. Finally, we calculated the correlation between each RNA quality index and the amount of library product (1(st) PCR product per input RNA). The DV200 measure showed stronger correlation with the amount of library product than the RINe (R2=0.8208 for the DV200 versus 0.6927 for the RINe). Receiver operating characteristic curve analyses revealed that the DV200 was the better marker for predicting efficient library production than the RINe using a threshold of >10 ng/ng for the amount of the 1(st) PCR product per input RNA (cutoff value for the RINe and DV200, 2.3 and 66.1%; area under the curve, 0.99 and 0.91; sensitivity, 82% and 92%; and specificity, 93% and 100%, respectively). Our results indicate that NGS libraries prepared using RNA samples with the DV200 value>66.1% exhibit greater sensitivity and specificity than those prepared with the RINe values>2.3. These findings suggest that the DV200 is superior to the RINe, especially for low-quality RNA, because it is a more consistent assessment of the amount of the 1(st) NGS library product per input