Media 1: Dynamic autofluorescence imaging of intracellular components inside living cells using direct electron beam excitation

Originally published in Biomedical Optics Express on 01 February 2014 (boe-5-2-378

Media 1: Dynamic and high-resolution live cell imaging by direct electron beam excitation

Originally published in Optics Express on 27 February 2012 (oe-20-5-5629

Comparisons between transcriptomes deduced by different assemblers.

<p>Scatter plot graphs show the length distribution of <i>IS</i>-transcripts deduced by Trinity (blue), Trans-ABySS (magenta) and Velvet-Oases (green). As suggested by the total number and N50 length of <i>IS</i>-transcripts, Velvet-Oases tended to give long <i>IS</i>-transcripts at the expense of their total number, while Trans-ABySS deduces a large number of short <i>IS</i>-transcripts. Trinity was intermediate with respect to these parameters. The Venn diagram shows how much one assembler covers the <i>IS</i>-transcripts deduced by the other assemblers. The number of <i>IS</i>-transcripts is given in parentheses. Trinity covered almost all the <i>IS</i>-transcripts deduced by either Velvet-Oases or Trans-ABySS.</p

Functional annotation.

<p><b>A.</b> Summary of annotation. <b>B.</b> E-value-, similarity (% identity)- and species-distribution in NR annotation. Species-distribution indicates that many of the <i>C. pyrrhogaster IS</i>-transcripts are close to genes of amniotes as well as those of amphibians such as <i>Xenopus (Silurana)</i> and <i>Xenopus laevis</i>, rather than fishes (e.g., <i>Danio rerio</i>, 2.9%; <i>Oryzias latipes</i>, 1.9%; these are included in ‘<i>other</i>’). Interestingly, within amphibians, the newt seems to adhere to <i>X. (Silurana)</i> rather than to <i>X. laevis</i>.</p

Comparisons with other newt transcriptomes.

<p>Black circles: <i>IS</i>-transcripts of this study. Blue circles: cDNAs and EST-contigs reported in <i>C. pyrrhogaster</i>. Red circles: cDNAs and EST-contigs reported in <i>P. waltl</i>. Green circles: cDNAs, EST-contigs and <i>IS</i>-transcripts reported in <i>N. viridescens</i>. The values in each circle (written in corresponding color) mean the number and ratio of <i>IS</i>-transcripts, cDNAs or EST-contigs.</p

Validation by qPCR.

<p><b>A.</b> Workflow of sample preparations for qPCR. RPE-choroid tissues harvested from the right eyes (intact eye) of 5 retinectomized animals at day-10 or day-14 were used for the normal RPE, i.e., the day-0 (Stage E-0) sample. RPE-derived cells harvested together with the choroid from the left eyes (retinectomized eye) of the same animals were used for the day-10 (Stage E-1) or day-14 (Stage E-2) sample. 3 different day samples (day-0, -10 and -14) were randomly grouped as one set of samples for qPCR. For more details, see Methods. <b>B.</b> qPCR analysis. 20 selected genes which have been suggested or inferred to be regulated in an early phase of retinal regeneration were found in the current <i>in silico</i> transcriptomes. Changes in their relative expression level until day-14 po were examined. Results represent means and SE. The number in parenthesis indicates the number of independent sample sets (see Methods) except for <i>Pax6</i> and <i>Chx10</i>, whose expression was detected in 2 of 8 sample sets and 4 of 5 sample sets, respectively at day-14 po only. Statistical significance based on Sheffe’s test following the Friedman test (*: <i>p</i><0.05; **: <i>p</i><0.01), except for <i>Pax6</i> and <i>Chx10</i>.</p

A Transcriptome for the Study of Early Processes of Retinal Regeneration in the Adult Newt, <i>Cynops pyrrhogaster</i>

<div><p>Retinal regeneration in the adult newt is a useful system to uncover essential mechanisms underlying the regeneration of body parts of this animal as well as to find clues to treat retinal disorders such as <i>proliferative vitreoretinopathy</i>. Here, to facilitate the study of early processes of retinal regeneration, we provide a <i>de novo</i> assembly transcriptome and inferred proteome of the Japanese fire bellied newt (<i>Cynops pyrrhogaster</i>), which was obtained from eyeball samples of day 0–14 after surgical removal of the lens and neural retina. This transcriptome (237,120 <i>in silico</i> transcripts) contains most information of cDNAs/ESTs which has been reported in newts (<i>C. pyrrhogaster</i>, <i>Pleurodeles waltl</i> and <i>Notophthalmus viridescence</i>) thus far. On the other hand, <i>de novo</i> assembly transcriptomes reported lately for <i>N. viridescence</i> only covered 16–31% of this transcriptome, suggesting that most constituents of this transcriptome are specific to the regenerating eye tissues of <i>C. pyrrhogaster</i>. A total of 87,102 <i>in silico</i> transcripts of this transcriptome were functionally annotated. Coding sequence prediction in combination with functional annotation revealed that 76,968 <i>in silico</i> transcripts encode protein/peptides recorded in public databases so far, whereas 17,316 might be unique. qPCR and Sanger sequencing demonstrated that this transcriptome contains much information pertaining to genes that are regulated in association with cell reprogramming, cell-cycle re-entry/proliferation, and tissue patterning in an early phase of retinal regeneration. This data also provides important insight for further investigations addressing cellular mechanisms and molecular networks underlying retinal regeneration as well as differences between retinal regeneration and disorders. This transcriptome can be applied to ensuing comprehensive gene screening steps, providing candidate genes, regardless of whether annotated or unique, to uncover essential mechanisms underlying early processes of retinal regeneration.</p></div

CDS prediction.

<p>Blastx predicted CDSs in 71,511 <i>IS</i>-transcripts (for the length distribution, see the graph ‘Blast’; for protein/peptide sequences, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109831#pone.0109831.s005" target="_blank">Table S5</a>) while ESTscan predicted CDSs in 22,773 <i>IS</i>-transcripts (for the length distribution, see the graph ‘ESTScan’; for protein/peptide sequences, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109831#pone.0109831.s006" target="_blank">Table S6</a>).</p

Workflow from retinectomy to <i>de novo</i> assembly.

<p><b>A.</b> Retinectomy. <b>B.</b> Sample collection, mRNA-seq and <i>de novo</i> assembly. <i>Stage E-0</i>: The RPE immediately after retinectomy. <i>Stage E-1</i>: Almost all RPE cells that have lost their epithelial characteristics and formed aggregates have entered the S-phase of the cell-cycle. <i>Stage E-2</i>: Partially depigmented cells are segregated into two rudiment layers (pro-NR and pro-RPE), which give rise to a new neural retina and the RPE layer itself. Under the current experimental conditions, regenerating retinas at Stage E-1 and E-2 are obtained at day-10 and -14 po <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109831#pone.0109831-Chiba1" target="_blank">[8]</a>.</p

GO classification.

<p>GO classification.</p