Merging multiple sets of loci.

<p>SeaSight offers several methods to combine locus data from different sources. Strong horizontal lines represent genomic loci, fine lines represent reads in the top panel. See text for details.</p

Transformation matrix for part of the case study (using three Affymetrix CEL files and three sequencing result files for the same kidney sample [<b>21</b>]).

<p>Experiments are displayed as rows, transformations as boxes with color indicating grouping of the transformations. Final data properties are displayed on the right side. Transformations can be added, removed and configured using context menus.</p

Functional analysis of <i>cg129</i>5 and <i>cg0548</i> by knockdown experiments.

<p>(A, B) <i>In vi</i>vo imaging of an siRNA-injected embryo (200 µM Stealth-RNA, 1-cell stage microinjection) imaged at 6 hpi (A) and 6,5 hpi (B). The white arrow in (A) indicates the site of the missing micromere cell after incomplete third cleavage. The same embryo was imaged 30 min later (6,5 hpi, B). The white arrow indicates the micromere cell that has just formed after a delayed division of one blastomere cell from the 4-cell stage. The changing shape of the other blastomeres at 6,5 hpi is a sign of initiated fourth cleavage (from 8- to 16-cell stage). Scale bar corresponds to 100 µm. (<b>C</b>) A ventro-lateral view of a DAPI-stained, control morpholino injected embryo fixed at 5 dpi. The white arrows indicate the position of the midgut anlagen (disc-shape structures). The ectodermal cell rows are clearly visible on the posterior ventral side of the embryo. (<b>D</b>) A ventro-lateral DAPI view of a anti-<i>cg0548</i> morpholino-injected embryo fixed at 5 dpi. The white arrow indicates the single disc-shape structure in the anterior part, whereas the second one is missing. (<b>E</b>) Phenotype scoring and survival of control and anti-<i>cg0548</i> morpholino-injected embryos at 5 dpi. More than 80% of the control embryos show normal germ band formation. In contrast, only 15% of the anti-<i>cg0548</i> morpholino-injected embryos were developing normal and 30% were already dead at the observed time point. (<b>F</b>) The <i>cg0548</i> RNA is coding for a small glycine-rich protein. The protein sequence includes 11 tandem repeats of the sequence Ala-Val-Gly-Gly-Gly-Ser-Gly-Phe-Gln-Pro (AVGGGSGFQP). Possible homologs of Ph-cg0548 were found in a wide range of organisms (from the closely related <i>Daphnia pulex</i> to the worm <i>Caenorhabditis elegans</i>). These proteins have short amino acid sequence (several hundred residues) and glycine-rich tandem repeats. Alignment of the repetitive element shows that <i>Parhyale</i> and <i>Daphnia</i> share the highest similarity, including a serine and an aromatic residue (F/Y). The position of the proline residue is shared between <i>Parhyale</i> and <i>C. elegans</i>. (<b>G</b>) Sequence alignment of the non-repetitive N-terminal part of the protein shows that <i>C. elegans</i> and <i>Parhyale</i> both have a stretch of aliphatic residues and a conserved phenylalanine, followed by a threonine.</p

The Maternal Transcriptome of the Crustacean <em>Parhyale hawaiensis</em> Is Inherited Asymmetrically to Invariant Cell Lineages of the Ectoderm and Mesoderm

<div><h3>Background</h3><p>The embryo of the crustacean <em>Parhyale hawaiensis</em> has a total, unequal and invariant early cleavage pattern. It specifies cell fates earlier than other arthropods, including <em>Drosophila</em>, as individual blastomeres of the 8-cell stage are allocated to the germ layers and the germline. Furthermore, the 8-cell stage is amenable to embryological manipulations. These unique features make <em>Parhyale</em> a suitable system for elucidating germ layer specification in arthropods. Since asymmetric localization of maternally provided RNA is a widespread mechanism to specify early cell fates, we asked whether this is also true for <em>Parhyale</em>. A candidate gene approach did not find RNAs that are asymmetrically distributed at the 8-cell stage. Therefore, we designed a high-density microarray from 9400 recently sequenced ESTs (1) to identify maternally provided RNAs and (2) to find RNAs that are differentially distributed among cells of the 8-cell stage.</p> <h3>Results</h3><p>Maternal-zygotic transition takes place around the 32-cell stage, i.e. after the specification of germ layers. By comparing a pool of RNAs from early embryos without zygotic transcription to zygotic RNAs of the germband, we found that more than 10% of the targets on the array were enriched in the maternal transcript pool. A screen for asymmetrically distributed RNAs at the 8-cell stage revealed 129 transcripts, from which 50% are predominantly expressed in the early embryonic stages. Finally, we performed knockdown experiments for two of these genes and observed cell-fate-related defects of embryonic development.</p> <h3>Conclusions</h3><p>In contrast to <em>Drosophila</em>, the four primary germ layer cell lineages in <em>Parhyale</em> are specified during the maternal control phase of the embryo. A key step in this process is the asymmetric distribution of a large number of maternal RNAs to the germ layer progenitor cells.</p> </div

Immunodetection of early embryonic transcription.

<p>Immunohistochemical detection of active RNAPII was performed with a commercially available monoclonal antibody against the Ser2 phosphorylated CTD of RNAPII (H5) and an Alexa 446 conjugated secondary antibody. (A-C) DAPI stained nuclei of a 16-cell stage embryo (A), 32-cell stage embryo (B) and a 100-cell stage embryo (C). An antibody signal was not detected in 16-cell stage embryos (A′). First signs of active transcription were visible in 32-cell stage embryos (B′) and fully active transcription was detected in 100-cell stage embryos (C′). Scale bar corresponds to 100 µm. In order to confirm the whole mount immunostainings, we performed a Western blot with protein extracts from 1-cell, 8-cell and 100-cell embryos (<b>D</b>). The 220 kDa phosphorylated RNAPII was detected only in the 100-cell stage protein extract (<b>D</b>′).</p

Microarray analysis of <i>Parhyale</i> embryos.

<p>Labelled RNA pools from transcriptionally inactive embryos (1- to 4-cell stage) and RNA pools from transcriptionally active embryos (germ band and limb bud stages) were hybridized together in a two-colour microarray experiment. (A) The MA plot uses the logarithmic fold-change value “M” as the y-axis and the logarithmic average expression value “A” as the x-axis, where: M = log2(zyg/mat), A = 1/2(log2(mat)+log2(zyg)), mat = signal intensity for transcriptionally inactive embryos, zyg = signal intensity for transcriptionally active embryos. The red data points represent the 690 unique maternally enriched RNAs, which constitute more than 10% of all unique gene sequences on the microarray. (B, C) The 8-cell embryo consists of four macromeres and four micromeres, each of them invariantly contributing to only one of the germ layers or the germ line. The three blue-coloured blastomeres give rise to ectoderm, the green-coloured blastomeres establish mesoderm, the yellow-coloured micromere is the progenitor of endoderm and the red-coloured cell contributes to the germ line. Blastomeres of the 8-cell stage were separated from each other by dissection and used for preparation of labelled cRNA for a two-coloured microarray experiment. The comparison between RNA from mesoderm progenitors and the rest of the 8-cell stage embryo (progenitors for ectoderm, endoderm and germline) resulted in the identification of a total of 129 asymmetrically localized RNAs. In the first experiment we found 35 RNAs overrepresented in the mesoderm progenitors transcript pool and 40 RNAs that were underrepresented. The second experiment approach revealed 33 transcripts enriched in ectoderm progenitors and 31 others that were predominantly found in the RNA pool from mesoderm, endoderm and germline progenitors. The four groups of asymmetrically localized RNAs from the two experiments add up to 139 because 10 of the RNAs were found in both experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056049#pone.0056049.s007" target="_blank">Table S4</a>).</p

Asymmetrically distributed RNAs at the 8-cell stage of <i>Parhyale</i>.

<p>(A) GO enrichment analysis for the asymmetrically distributed RNAs at the 8-cell stage of <i>Parhyale</i>, as well as for the maternally provided transcripts. The bars represent the GO-term fold-overrepresentation (percent of sequences in the analysed group associated to a certain GO-term over the percentage for this GO-term in the reference group) for significantly overrepresented GO-terms (Fisher’s Exact Test P-value less than 0.05) within the 690 maternally enriched targets (red bars), the 35 RNAs enriched in mesoderm progenitors (green bars) and the 33 ectoderm progenitor transcripts (blue bars). (B) A cross-correlation was performed between the data sets for (1) asymmetrically distributed RNAs at the 8-cell stage and (2) differentially expressed genes in transcriptionally inactive (pre-MZT) and transcriptionally active embryos. The left circle of the Venn diagram contains the 690 maternally enriched RNAs, 65 of which are asymmetrically distributed at the 8-cell stage (intersection with lower circle). The intersection of the left and right circles includes 236 targets expressed at equal levels during early and late embryonic development, with 12 being asymmetrically localized at the 8-cell stage. The right circle consists of the 568 genes predominantly expressed from the zygotic genome at later embryonic stages, including the 29 asymmetric at the 8-cell stage. There are 35 low-expressed RNAs, which are still showing statistically significant changes between the blastomeres of the 8-cell.</p

MOESM1 of Immune monitoring and TCR sequencing of CD4 T cells in a long term responsive patient with metastasized pancreatic ductal carcinoma treated with individualized, neoepitope-derived multipeptide vaccines: a case report

Additional file 1: Figure S1. Work flow of immune monitoring techniques

MOESM5 of Immune monitoring and TCR sequencing of CD4 T cells in a long term responsive patient with metastasized pancreatic ductal carcinoma treated with individualized, neoepitope-derived multipeptide vaccines: a case report

Additional file 5: Figure S5. Peptide-responsive clones at IM3 all displayed an CD45RA+ effector memory TEMRA phenotype. One representative example (clone #54) is shown in (a). In contrast, clones obtained from control cultures without peptide stimulation expressed a CD45RAneg effector memory phenotype, data by clone #6 are shown in (b)

Additional file 3: Figure S1. of O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis

Calibration curve of the novel O5-S qPCR. (DOCX 22 kb