YCZ-18 is a new brassinosteroid biosynthesis inhibitor.

Plant hormone brassinosteroids (BRs) are a group of polyhydroxylated steroids that play critical roles in regulating broad aspects of plant growth and development. The structural diversity of BRs is generated by the action of several groups of P450s. Brassinazole is a specific inhibitor of C-22 hydroxylase (CYP90B1) in BR biosynthesis, and the application use of brassinazole has emerged as an effective way of complementing BR-deficient mutants to elucidate the functions of BRs. In this article, we report a new triazole-type BR biosynthesis inhibitor, YCZ-18. Quantitative analysis the endogenous levels of BRs in Arabidopsis indicated that YCZ-18 significantly decreased the BR contents in plant tissues. Assessment of the binding affinity of YCZ-18to purified recombinant CYP90D1 indicated that YCZ-18 induced a typical type II binding spectrum with a Kd value of approximately 0.79 μM. Analysis of the mechanisms underlying the dwarf phenotype associated with YCZ-18 treatment of Arabidopsis indicated that the chemically induced dwarf phenotype was caused by a failure of cell elongation. Moreover, dissecting the effect of YCZ-18 on the induction or down regulation of genes responsive to BRs indicated that YCZ-18 regulated the expression of genes responsible for BRs deficiency in Arabidopsis. These findings indicate that YCZ-18 is a potent BR biosynthesis inhibitor and has a new target site, C23-hydroxylation in BR biosynthesis. Application of YCZ-18 will be a good starting point for further elucidation of the detailed mechanism of BR biosynthesis and its regulation

eVIDENCE: a practical variant filtering for low-frequency variants detection in cell-free DNA

血中遊離DNAの高精度解析手法を開発 --リキッドバイオプシーによるゲノム医療へ--. 京都大学プレスリリース. 2019-10-29.Plasma cell-free DNA (cfDNA) testing plays an increasingly important role in precision medicine for cancer. However, circulating cell-free tumor DNA (ctDNA) is highly diluted by cfDNA from non-cancer cells, complicating ctDNA detection and analysis. To identify low-frequency variants, we developed a program, eVIDENCE, which is a workflow for filtering candidate variants detected by using the ThruPLEX tag-seq (Takara Bio), a commercially-available molecular barcoding kit. We analyzed 27 cfDNA samples from hepatocellular carcinoma patients. Sequencing libraries were constructed and hybridized to our custom panel targeting about 80 genes. An initial variant calling identified 36, 500 single nucleotide variants (SNVs) and 9, 300 insertions and deletions (indels) across the 27 samples, but the number was much greater than expected when compared with previous cancer genome studies. eVIDENCE was applied to the candidate variants and finally 70 SNVs and 7 indels remained. Of the 77 variants, 49 (63.6%) showed VAF of < 1% (0.20–0.98%). Twenty-five variants were selected in an unbiased manner and all were successfully validated, suggesting that eVIDENCE can identify variants with VAF of ≥ 0.2%. Additionally, this study is the first to detect hepatitis B virus integration sites and genomic rearrangements in the TERT region from cfDNA of HCC patients. We consider that our method can be applied in the examination of cfDNA from other types of malignancies using specific custom gene panels and will contribute to comprehensive ctDNA analysis

YCZ-18-treated plants display the BR-deficient phenotype.

<p><b>YCZ-18</b>-treated plants (0.3, 1, 3 μM), Brz220-treated plants (3 μM) and brassinosteroid-deficient mutant (<i>det2</i>) plants were grown for 6 days in the dark (A) and for 10 days in the light (B-G) on medium containing the chemical indicated. The control plants (Cont) were untreated. Scale bar = 5 mm.</p

YCZ18-treated Arabidopsis in response to cathasterone (CT) and teasterone (TE).

<p>Five-day-old <i>Arabidopsis</i> seedlings (8A, 8E, white bar), treated with 0.5 μM <b>YCZ-18</b> (8B, 8E, yellow bar), treated with 0.5 μM <b>YCZ-18</b> together with 30 μM cathasterone (CT) (8C, 8E, red bar), or treated with 0.5 μM <b>YCZ-18</b> together with 10 μM teasterone (TE) (8D, 8E, blue bar). Data are the means ± s.e. obtained from 30 seedlings. Scale bar = 3 mm.</p

YCZ-18 regulates the expression of BR-responsive genes.

<p>Quantitative RT-PCR experiment measuring the relative expression levels of a BR-upregulated gene (<i>TCH4</i>) (A), a BR biosynthetic gene (<i>DWF4</i>) (B) and two photosynthesis genes (<i>LHCP</i> (C) and <i>rbcS</i> (D)) of the wild-type plant (Cont), <b>YCZ-18</b>-treated (0.3, 1, 3 μM), Brz-treated (3 μM) and brassinosteroid-deficient mutant <i>det2</i>. Plants were grown for 10 days in the light (A, B) and for 6 days in the dark (C, D) on a medium containing the chemical indicated. All results are means ± s.e.</p

Binding of YCZ-18 to CYP90D1.

<p>Absorption spectra of oxidized CYP90D1 (blue line) and its <b>YCZ-18</b> complex (pink line). Recombinant CYP90D1 (3.5 μM) was dissolved in 50 mM NaH₂PO₄ (pH 7.0) with 0.1% Tween 20 containing 20% glycerol, and <b>YCZ-18</b> was added to CYP90D1 at a final concentration of 16 μM (A). Spectrophotometric titration of CYP90D1 with <b>YCZ-18</b> induced spectral changes in CYP90D1. <b>YCZ-18</b> was added to CYP90D1 (3.5 μM) at various final concentrations (a, 0.7; b, 1; c, 2; d, 4; e, 8; f, 12; g, 16 μM) (B). The spectral dissociation constant was calculated from a double reciprocal plot of absorbance differences, ΔA (436–415 nm) versus the <b>YCZ-18</b> concentrations given 0.79 μm (C). The experiment was duplicated to establish reproducibility.</p

Effect of YCZ-18 on the growth of Arabidopsis in soil.

<p>The application of <b>YCZ-18</b> on wild-type <i>Arabidopsis</i> was performed by spraying an aqueous solution of <b>YCZ-18</b> (5 μM) onto ten-day-old wild-type <i>Arabidopsis</i> plants (approximately 0.2 pmol/plant), as indicated in the methods section. Four-week-old <i>Arabidopsis</i> seedlings (A), four-week-old <i>Arabidopsis</i> treated with <b>YCZ-18</b> (B), six-week-old <i>Arabidopsis</i> (C), six-week-old <i>Arabidopsis</i> seedlings treated with <b>YCZ-18</b> (D), rosette leaf number of six-week-old <i>Arabidopsis</i> at bolting from three plants (E). Data are the means ± s.e. obtained from 3 plants. Scale bar = 1 cm.</p

Effect of YCA-18 on the hydroponic growth of <i>Arabidopsis</i>.

<p><i>Arabidopsis</i> plants grown under hydroponic conditions with or without <b>YCZ-18</b> treatment were treated as indicated in the methods section. Forty-five-day-old <i>Arabidopsis</i> (A); forty-five-day-old <i>Arabidopsis</i> treated with <b>YCZ-18</b> at 0.1 μM (B), 0.5 μM (C), or 1 μM (D). The growth curves of <i>Arabidopsis</i> treated with different concentrations of <b>YCZ-18</b> (E). Data are the means ± s.e. obtained from 8 to 10 plants. Bar = 1 cm.</p