Additional file 1 of Synergetic delivery of artesunate and isosorbide 5-mononitrate with reduction-sensitive polymer nanoparticles for ovarian cancer chemotherapy

Additional file 1. Additional information

Edible Safety Assessment of Genetically Modified Rice T1C-1 for Sprague Dawley Rats through Horizontal Gene Transfer, Allergenicity and Intestinal Microbiota

<div><p>In this study, assessment of the safety of transgenic rice T1C-1 expressing Cry1C was carried out by: (1) studying horizontal gene transfer (HGT) in Sprague Dawley rats fed transgenic rice for 90 d; (2) examining the effect of Cry1C protein in vitro on digestibility and allergenicity; and (3) studying the changes of intestinal microbiota in rats fed with transgenic rice T1C-1 in acute and subchronic toxicity tests. Sprague Dawley rats were fed a diet containing either 60% GM <i>Bacillus thuringiensis</i> (Bt) rice T1C-1 expressing Cry1C protein, the parental rice Minghui 63, or a basic diet for 90 d. The GM Bt rice T1C-1 showed no evidence of HGT between rats and transgenic rice. Sequence searching of the Cry1C protein showed no homology with known allergens or toxins. Cry1C protein was rapidly degraded <i>in vitro</i> with simulated gastric and intestinal fluids. The expressed Cry1C protein did not induce high levels of specific IgG and IgE antibodies in rats. The intestinal microbiota of rats fed T1C-1 was also analyzed in acute and subchronic toxicity tests by DGGE. Cluster analysis of DGGE profiles revealed significant individual differences in the rats' intestinal microbiota.</p></div

Safety assessment of HGT.

<p>PCR analysis of <i>cry1C</i> gene from masseter muscle (<b>a</b>), duodenum (<b>b</b>), ileum (<b>c</b>). M, DL2000 marker; 1–3, genomic DNA of T1C-1, Minghui 63 and control group, respectively; 4–6, <i>prl</i> of the genomic DNA of T1C-1, Minghui 63 and control group, respectively; 7, 35S promoter positive control; 8–10, 35S promoter of T1C-1, Minghui 63 and control group, respectively; 11, NOS terminator positive; 12–14, NOS terminator of T1C–1, Minghui 63 and control group, respectively; 15, exogenous gene control; 16–18, exogenous gene <i>cry1C</i> of T1C-1, Minghui 63 and control group, respectively. PCR analysis of <i>cry1C</i> gene from microbes in anaerobic cultures (<b>d</b>), <i>Salmonella</i> (<b>e</b>), <i>Lactobacilli</i> (<b>f</b>), <i>Streptococcus</i> (<b>g</b>), <i>E</i>. <i>coli</i> (<b>h</b>). M, DL2000 marker; 1–3, genomic DNA of T1C-1, Minghui 63 and control group, respectively; 4–6, bacterial 16S DNA of T1C-1, Minghui 63 and control group, respectively; 7, 35S promoter positive control; 8–10, 35S promoter of T1C-1, Minghui 63 and control group, respectively; 11, NOS terminator positive; 12–14, NOS terminator of T1C–1, Minghui 63 and control group, respectively; 15, exogenous gene control; 16–18, exogenous gene <i>cry1C</i> of T1C-1, Minghui 63 and control group, respectively.</p

Shannon index (H) and Simpson’s index (D) of each band from DGGE bands and phylogenetic tree analysis of the 16S rDNA gene sequences of the obtained clones.

<p><b>(a,b,c)</b> A1–A6, B1–B6 and C1–C6 have the same meanings as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163352#pone.0163352.g004" target="_blank">Fig 4</a>, ■The value of H, ▲ the value of D. (<b>d</b>) GenBank accession numbers are given in parentheses. The scale bar indicates the 0.05 evolutionary distance unit. Bootstrap values (percent) are shown at branch nodes.</p

DGGE profiles and cluster analysis of the profiles from rat fecal bacteria.

<p>(<b>a</b>,<b>b</b>) DGGE profiles in test group fed daily with Bt rice T1C-1 expressing Cry1C. (<b>c</b>,<b>d</b>) Cluster analysis of DGGE profiles of a, b. (<b>e,f</b>) DGGE profiles in control group fed with rice Minghui 63. (<b>g,h</b>) Cluster analysis of DGGE profiles of g, h. (<b>i</b>,<b>j</b>) DGGE profiles in control group fed with untreated food. (<b>k,l</b>) Cluster analysis of DGGE profiles of i, j. <b>a</b>, 1–6, samples from test group A1; 7–12, samples from A2; 13–18, samples from A3 at 0, 30, 60, 80, 85 and 90 d after feeding with transgenic rice T1C-1 expressing Cry1C. <b>b</b>, 1–6, samples from A4; 7–11, samples from A5; 12–18, samples from A6 at 0, 30, 60, 80, 85 and 90 d after feeding with transgenic rice T1C-1 expressing Cry1C. <b>e</b>: 1–6: samples from control group B1; 7–12: samples from B2; 13–18: samples from B3 at 0, 30, 60, 80, 85 and 90 d after feeding with rice Minghui 63; <b>f</b>: 1–6: samples from B4; 7–12: samples from B5; 13–18: samples from B6 at 0, 30, 60, 80, 85 and 90 d after feeding with rice Minghui 63. <b>i</b>: 1–6: samples from control group fed untreated food daily C1; 7–12: samples from C2; 13–18: samples from C3 at 0, 30, 60, 80, 85 and 90 d after feeding with untreated food daily; <b>j</b>: 1–6: samples from C4; 7–12: samples from C5; 13–18: samples from C6 at 0, 30, 60, 80, 85 and 90 d after feeding with untreated food daily.</p

SDS-PAGE, western blot analysis of Cry1C protein and the protein degradation test in SGF and SIF.

<p>(<b>a</b>) M, molecular mass protein standards; 1–3, lysis supernatant of Transetta (DE3) cells, cells transformed with pET30a (+) and pET30a–Cry1C, respectively; 4, 5, Cry1C fusion protein. (<b>b</b>) Western blot analysis of Cry1C protein expressed in <i>E</i>. <i>coli</i> (lane 1) and the rice-derived Cry1C protein (lane 2). Gel staining (<b>c,e</b>) and western blot analysis (<b>d</b>,<b>f</b>) of fusion protein digested with simulated gastric fluid (SGF), simulated intestinal fluid (SIF), respectively. M, molecular mass protein standards; 1–8, 0 s, 15 s, 30 s, 60 s, 2 min, 10 min, 30 min and 60 min after digestion, respectively.</p

DGGE profiles and analysis by UPGAMA from rat fecal bacteria in the control and test group, respectively.

<p>(<b>a</b>) 1–4, 5–8, 9–12, samples from R1, R2 and R3 (the three rats in the control group) at 0, 1, 7 and 14 d after gavage respectively. (<b>b</b>) 1–4, 5–8, 9–12, samples from T1, T2 and T3 (the three rats in the test group) at 0, 1, 7 and 14 d, respectively. (<b>c</b>) 1–4, 5–8, 9–12, Samples from T4, T5, T6 (the other three rats in the test group) at 0, 1, 7 and 14 d, respectively. (<b>d–f</b>) analysis of rat fecal samples by UPGAMA after gavage.</p