Paclitaxel-Loaded <i>N</i>‑Octyl‑<i>O</i>‑sulfate Chitosan Micelles for Superior Cancer Therapeutic Efficacy and Overcoming Drug Resistance

The nanoparticle-based drug delivery system holds great attraction to overcome or circumvent multidrug resistance (MDR) in cancer to date. In this work, a synthesized amphiphilic graft copolymer, <i>N</i>-octyl-<i>O</i>-sulfate chitosan (NOSC), and its paclitaxel (PTX)-encapsulated micelles (PTX-M) have been systematically investigated on the MDR reversal effect <i>in vitro</i> and <i>in vivo</i> as well as the mechanism of P-glycoprotein (P-gp) inhibition. NOSC in a wide concentration range even above the critical micelle concentration showed an effective effect on inhibiting P-gp-mediated PTX efflux, which was remarkably different from the surfactants and the Pluronic copolymers. Multiple mechanisms were involved in this effect of NOSC, such as stimulating P-gp ATPase, competitively impeding the binding of PTX with P-gp and reducing the fluidity of the cell membrane. PTX-M presented the highest cellular uptake and the lowest efflux rate of PTX, thereby yielding the optimal cytotoxicity on both the human hepatocellular liver carcinoma (HepG2) cells and the multidrug resistance HepG2 (HepG2-P) cells, which resulted from a combination of the inhibiting P-gp effect of NOSC and the bypassing P-gp action of the intact PTX-M. Additionally, PTX-M had superior blood persistence, tumor accumulation, and therapeutic efficacy after intravenous injection into the tumor-bearing mice. Furthermore, it was demonstrated that most of PTX-M as an intact form was delivered at the tumor site, which ensures the synergetic effect of NOSC micelles on drug delivery and P-gp inhibition. The aforementioned results suggested that NOSC micelles presented promising potential as an anticancer drug carrier for enhanced MDR cancer therapy

A Potential Role for CHH DNA Methylation in Cotton Fiber Growth Patterns

<div><p>DNA methylation controls many aspects of plant growth and development. Here, we report a novel annual growth potential change that may correlate with changes in levels of the major DNA demethylases and methyltransferases in cotton ovules harvested at different times of the year. The abundances of DNA demethylases, at both the mRNA and protein levels, increased significantly from February to August and decreased during the remainder of the 12-month period, with the opposite pattern observed for DNA methyltransferases. Over the course of one year, substantial changes in methylcytosine content was observed at certain CHH sites (H = A, C, or T) in the promoter regions of the <i>ETHYLENE RESPONSIVE FACTOR 6</i> (<i>ERF6</i>), <i>SUPPRESSION OF RVS 161 DELTA 4</i> (<i>SUR4</i>) and <i>3-KETOACYL-COA SYNTHASE 13</i> (<i>KCS13</i>), which regulate cotton fiber growth. Three independent techniques were used to confirm the annual fluctuations in DNA methylation. Furthermore, in homozygous RNAi lines specifically targeting REPRESSOR OF SILENCING 1 (ROS1, a conserved DNA demethylase domain), promotion of DNA methylation significantly reduced fiber growth during August.</p> </div

Methylation-sensitive endonuclease digested PCR and Southern analysis of <i>ERF6</i>, <i>SUR4</i>, and <i>KCS13</i> upstream regions in ^ROS1RNAi lines.

<p>(A) Analysis of relative <i>ERF6</i> transcription in ovules from ^ROS1RNAi lines by qRT-PCR. The level of <i>ERF6</i> transcripts in ovules from the empty vector line (V) was arbitrarily defined as 1. (B) Southern blot analysis of genomic DNA prepared from ^ROS1RNAi lines digested thoroughly with <i>BstX</i>I. See detailed information in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060547#pone-0060547-g004" target="_blank">Figure 4</a> legend. Similar qRT-PCR experiments were performed for <i>SUR4</i> (C) and <i>KCS13</i> (E) transcriptions, as well as similar Southern experiments for <i>SUR4</i> (D) and <i>KCS13</i> (F), respectively. Note the reduced intensities of the <i>BstX</i>I-, <i>HinF</i>I- and <i>Bsl</i>I-cleaved bands in all three RNAi lines compared to the vector line.</p

Annual changes in DNA methylation patterns in cotton ovules.

<p>(A) qRT-PCR analysis of DNA methylation and demethylation genes reported in GO: 0006306. The <i>ACT7</i> transcript, normalized against that of all 20 housekeeping genes reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060547#pone.0060547.s009" target="_blank">Table S5</a>, was used as the internal standard. (B) Western blot analysis of DRM1/2 and ROS1. May, August, and November signal intensities were normalized to those from February (arbitrarily set to 1). Values (mean ± SE from three independent experiments) are shown beneath representative bands. Cotton UBQ was used as a loading control. (C) <i>KCS13</i>, <i>SUR4</i> and <i>ERF6</i> transcript levels changed over the course of the year, as quantified by qRT-PCR. May, August, and November values were normalized to February values (arbitrarily set to 1).</p

Bisulfite sequencing of <i>ERF6</i>, <i>SUR4</i>, and <i>KCS13</i> upstream regions over one year.

<p>The fragments to be examined were amplified by sequence-specific PCR primers after treating the template DNA with bisulfite. 17 unique “non-sister” individual clones from independent PCR reactions were selected for sequencing. Each line represents one unique “non-sister” individual bisulfite sequencing result. Only cytosines are shown using red for CG context, blue for CHG, and green for CHH. Open circles, unmethylated cytosines; closed circles, methylated cytosines; black triangles, cytosines showed annual methylation changes; red triangles, cytosine sites used for methylation-sensitive endonuclease digested PCR and Southern assay. The same designations were used for all bisulfite sequencing data reported in the current work. (A) The sequence from 272 to 662 nt in DQ464372 from <i>ERF6</i> upstream region was analyzed for DNA methylation. (B) The sequence from 322 to 646 nt in JQ922563 from <i>SUR4</i> upstream region was analyzed for DNA methylation. (C) The sequence from 1641 to 1939 nt in JQ922562 from <i>KCS13</i> upstream region was analyzed for DNA methylation.</p

Methylation-sensitive endonuclease digested PCR and Southern analysis of <i>ERF6</i>, <i>SUR4</i>, and <i>KCS13</i> upstream regions over one year.

<p>(A) Methylation-sensitive endonuclease digested PCR amplification of <i>ERF6</i> upstream region. Top: schematic diagram of the identification of a methylation-sensitive <i>BstX</i>I digenstion site (CCANNNNNNTGG) at −275 bp of the <i>ERF6</i> promoter. The bold C indicates a CHH site with annual methylation pattern change, corresponding to the cytosine labelled with red triangles in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060547#pone-0060547-g003" target="_blank">Figure 3A</a>. Bottom: PCR amplification using genomic DNA with (+) or without (−) <i>BstX</i>I digestion. (B) Southern blot of genomic DNA harvested at different times of the year, first digested by a methylation non-sensitive endonuclease <i>Mbo</i>II (TCTTC) to obtain a full length fragment of 605 bp from −621 to −15 of <i>ERF6</i> upstream regions, then digested thoroughly with <i>BstX</i>I, and probed with the fragment from −263 to −21 nt. The signal intensities of the band of <i>BstX</i>I-cleaved 244 bp changed at different time-of-year (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060547#pone.0060547.s012" target="_blank">Table S8</a>), indicating the methlytion levels of this CHH site were different, consistent with the bisulfite sequencing data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060547#pone-0060547-g003" target="_blank">Figure 3A</a> and methylation-sensitive endonuclease digested PCR results in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060547#pone-0060547-g004" target="_blank">Figure 4A</a>. The same methylation-sensitive endonuclease digested PCR experiments were performed for the upstream regions of <i>SUR4</i> (C) and <i>KCS13</i> (E), except the methylation-sensitive endonucleases used were <i>HinF</i>I and <i>Bsl</i>I, respectively. Further, the same methylation-sensitive endonuclease digested Southern experiments were performed for the upstream regions of <i>SUR4</i> (D) and <i>KCS13</i> (F), except the genomic DNA were first digested by <i>Bcl</i>I (TGATCA) and <i>NSi</i>I (ATGCAT), then digested by methylation-sensitive endonucleases <i>HinF</i>I (GANTC) and <i>Bsl</i>I (CCNNNNNNNGG), respectively. The signal intensities of <i>HinF</i>I- and <i>Bsl</i>I-cleaved 330 bp and 837 bp changed similarly (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060547#pone.0060547.s012" target="_blank">Table S8</a>).</p

Annual growth potential change of cotton plants.

<p>(A) Cotton ovule growth potential as a function of month in which ovules were harvested. Ovules were harvested 1 dpa during the month indicated, cultured for 6 d, and measured for fiber length. Numbers indicate fiber length (mean ± SE, in mm). Each ovule in this panel is a representative of thirty in the same culture. (B) Growth of cotton fibers from ovules harvested over the same monthly cycle for three consecutive years. (C) Cotton fiber length and first main stem internode length <i>in planta</i> in four different seasons. Error bars, SE. In (A–C), n = 6; In (A,C), *p<0.05, **p<0.01, ***p<0.001.</p

Bisulfite sequencing of <i>ERF6</i>, <i>SUR4</i>, and <i>KCS13</i> upstream regions in ^ROS1RNAi lines.

<p>The same primers were used for bisulfite treated PCR and sequencing as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060547#pone-0060547-g003" target="_blank">Figure 3</a>. (A), promoter region of <i>ERF6</i>; (B), promoter region of <i>SUR4</i>; (C), promoter region of <i>KCS13</i>; V, RNAi line with empty vector; R1–R3, RNAi line <i>ROS1-1</i> to <i>ROS1-3</i>. All symbols are same to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060547#pone-0060547-g003" target="_blank">Figure 3</a>.</p

MOESM5 of Systems analysis of phosphate-limitation-induced lipid accumulation by the oleaginous yeast Rhodosporidium toruloides

Additional file 5: Table S5. Model summaries for the discrimination between the Pi-replete and Pi-limited samples from LC-MS data for metabolomic analysis

Phenotype and genetic identification of ^ROS1RNAi lines.

<p>(A) Homozygous ^ROS1RNAi cotton lines at flowering. Vector plants carry the empty vector and showed identical properties with the parent. (B) Cotton ovules from RNAi lines that flowered during August (upper panel) or February (lower panel) were cultured for 6 d before being photographed for fiber measurement. (C) Analysis of <i>ROS1</i> transcripts in ovules from various ^ROS1RNAi lines by qRT-PCR (upper panel) and western blotting (lower panel).</p