mRNA <i>in situ</i> hybridization of <i>OsMTP11</i> in rice leaves.

<p><i>Blue</i> or <i>purple</i> precipitates indicate positive <i>OsMTP11</i> mRNA signals. <b>A.</b> Transverse sections of a mature leaf. <b>B.</b> Transverse sections of a young leaf bud. <b>C.</b> Longitudinal sections of a seedling leaf bud. <b>D.</b> Control with the transverse sections of leaf bud.</p

The <i>OsMTP11</i> putative promoter region (-2,250 bp) sequence.

<p>The transcription start site is denoted +1, and the putative start codon is underlined. Diagram of the <i>OsMTP11</i> promoter region using PlantCARE (<a href="http://bioinformatics.psb.ugent.be/webtools/plantcare/html/" target="_blank">http://bioinformatics.psb.ugent.be/webtools/plantcare/html/</a>) showed the presence of a number of potential cis-acting elements that respond to environmental signals. MRE, metal-response element [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174987#pone.0174987.ref034" target="_blank">34</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174987#pone.0174987.ref035" target="_blank">35</a>]; ABRE, abscisic acid-response element [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174987#pone.0174987.ref036" target="_blank">36</a>]; I-box, light-response element [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174987#pone.0174987.ref037" target="_blank">37</a>]; BS1EGCCR, "BS1 (binding site 1)" found in CCR gene promoter, which is a cis-element required for vascular expression of the cinnamoyl CoA reductase gene in <i>E</i>. <i>gunnii</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174987#pone.0174987.ref038" target="_blank">38</a>]. MREs include MRE1: 5’-TGCRCNC-3’ (R = A or G; N = any residue) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174987#pone.0174987.ref034" target="_blank">34</a>] and MRE2: 5’-HTHNNGCTGD-3’ (D = A, G, or T; H = A, C, or T; N = any residue) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174987#pone.0174987.ref035" target="_blank">35</a>].</p

Bioinformatics analyses of <i>OsMTP11</i> nucleotide and amino acid sequences.

<p><b>A.</b><i>OsMTP11</i> gene structure analysis in the Rice Genome Annotation Database (<a href="http://rice.plantbiology.msu.edu/" target="_blank">http://rice.plantbiology.msu.edu/</a>). <b>B.</b> Phylogenetic tree of the <i>MTP</i> family from rice and <i>Arabidopsis</i>. The tree was constructed using MEGA 4.0.2 by the neighbor-joining method. <i>Arabidopsis</i> MTP amino acid sequences were obtained from <a href="http://www.tigr.org" target="_blank">www.tigr.org</a>: AtMTP1, At2g46800; AtMTP2, At3g61940; AtMTP3, At3g58810; AtMTP4, At2g29410; AtMTP5, At3g12100; AtMTP6, At2g47830; AtMTP7, At1g51610; AtMTP8, At3g58060; AtMTP9, At1g79520; AtMTP10, At1g16310; AtMTP11, At2g39450; AtMTP12, At2g04620. Rice MTP amino acid sequences were downloaded from <a href="http://rice.plantbiology.msu.edu/" target="_blank">http://rice.plantbiology.msu.edu/</a>. OsMTP11, Os01g62070; OsMTP1, Os05g03780; OsMTP5, Os02g58580; OsMTP6, Os03g22550; OsMTP7, Os04g23180; OsMTP8, Os02g53490; OsMTP8.1, Os03g12580; OsMTP9, Os01g03914; OsMTP11, Os01g62070; OsMTP11.1, Os05g38670; OsMTP12, Os08g32680. <b>C.</b> Amino acid alignment of OsMTP11, AtMTP11 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174987#pone.0174987.ref011" target="_blank">11</a>] and ShMTP1 (AY181256) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174987#pone.0174987.ref012" target="_blank">12</a>]. Amino acid sequences of four predicted transmembrane (TM) segments are boxed. Amino acid residues with dark shading indicate conserved sequences, and residues with light gray shading indicate those conserved in two protein sequences. <b>D.</b> The predicted transmembrane helices of OsMTP11. The transmembrane domains were estimated using TMHMM2: <a href="http://www.cbs.dtu.dk/services/TMHMM/" target="_blank">www.cbs.dtu.dk/services/TMHMM/</a>. The peaks show the predicted transmembrane (TM) regions of proteins. These data indicate that OsMTP11 has four obvious TM regions.</p

Expression pattern of <i>OsMTP11</i> by real time RT-PCR.

<p><b>A.</b> Real time RT-PCR results of <i>OsMTP11</i> expression in wild-type rice plants (Nipponbare) from different tissues or organs. The amplification of the rice <i>OsUBQ5</i> (AK061988) gene was used as a control to normalize the transcript level of <i>OsMTP11</i>. <b>B.</b> Expression analysis of <i>OsMTP11</i> under different heavy metal stresses (Mn, Cd, Zn and Ni) by real time RT-PCR. The expression of <i>OsMTP11</i> is increased in rice roots and shoots treated with 0.5 mM CdCl₂, 5 mM Zn(NO₃)₂, 1 mM NiCl₂, 2 mM MnSO₄, 300 mM NaCl and 100 μM methyl viologen (MV) for different time periods. <b>C.</b> Expression analysis of <i>OsMTP11</i> under 300 mM NaCl and 100 μM methylviologen (MV) by real time RT-PCR.</p

List of <i>S</i>. <i>cerevisiae</i> mutant strains used in this research.

<p>Euroscarf (<a href="http://web.uni-frankfurt.de/fb15/mikro/euroscarf/" target="_blank">http://web.uni-frankfurt.de/fb15/mikro/euroscarf/</a>) is a collection of single-deletion mutants in <i>S</i>. <i>cerevisiae</i> (Frankfurt).</p

DNA methylation analysis of the <i>OsMTP11</i> promoter region.

<p><b>A.</b> Schematic distribution of CpG sites and CpG islands in the <i>OsMTP11</i> promoter region. Blue area indicates CpG islands. The scale bar indicates 100 bp. Red bar indicates the region analyzed by bisulfate sequencing. <b>B.</b> Comparison of percentages of cytosine methylation of three different types (CpG, CpHpG, and CpHpH) in rice seedling roots between control and under heavy metal stress (0.5 mM CdCl₂, 5 mM ZnSO_4, 2 mM MnSO_4, 1 mM NiCl₂) or salt treatment (300 mM NaCl) for 72 hours.</p

A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) Adjuvant for Hepatitis B Vaccine

<div><p>Although adjuvants are a common component of many vaccines, there are few adjuvants licensed for use in humans due to concerns about their toxic effects. There is a need to develop new and safe adjuvants, because some existing vaccines have low immunogenicity among certain patient groups. In this study, SBP, a hepatitis B surface antigen binding protein that was discovered through screening a human liver cDNA expression library, was introduced into hepatitis B vaccine. A good laboratory practice, non-clinical safety evaluation was performed to identify the side effects of both SBP and SBP-adjuvanted hepatitis B vaccine. The results indicate that SBP could enhance the HBsAg-specific immune response, thus increasing the protection provided by the hepatitis B vaccine. The safety data obtained here warrant further investigation of SBP as a vaccine adjuvant.</p></div

SBP could induce local irritation reaction.

<p>Representative photomicrographs of treated site are shown with original magnification: × 200 (HE staining). (a) NS group, 3 days after the last injection, normal tissue. (b) V+SBP (high dose) group, 3 days after the last injection, necrosis of myofiber (focal). (c) V+SBP (high dose) group, 3 days after the last injection, mesenchyme inflammatory cell infiltration and macrophage aggregation (multi-focus). (d) V+SBP (high dose) group, at the end of the recovery period, mesenchyme inflammatory cell infiltration and macrophage aggregation (multi-focus).</p

SBP adjuvanted hepatitis B vaccine could induce higher antibody titers and higher percentage of Th cells.

<p>Macaca fascicularis were immunized intramuscularly at days 1, 9, 15, 32 with vaccine alone (H) or together with SBP (H-S). (A) Blood samples were collected on indicated time points and HBsAg specific antibody titers were measured. (B)Percentage of CD4+ T cells was also measured at these time points. Results are expressed as the means ± SEM, * p<0.05.</p

Procedures of safety evaluation.

<p>(a) Experimental design of GLP non-clinical acute toxic test in ICR mice. (b) Experimental design of GLP non-clinical long toxic test in rats. (c) Experimental design of GLP non-clinical allergic test in guinea pigs.</p