Gene expression profiling of coelomic cells and discovery of immune-related genes in the earthworm, <i>Eisenia andrei</i>, using expressed sequence tags

<div><p>The coelomic cells of the earthworm consist of leukocytes, chlorogocytes, and coelomocytes, which play an important role in innate immunity reactions. To gain insight into the expression profiles of coelomic cells of the earthworm, <i>Eisenia andrei</i>, we analyzed 1151 expressed sequence tags (ESTs) derived from the cDNA library of the coelomic cells. Among the 1151 ESTs analyzed, 493 ESTs (42.8%) showed a significant similarity to known genes and represented 164 unique genes, of which 93 ESTs were singletons and 71 ESTs manifested as two or more ESTs. From the 164 unique genes sequenced, we found 24 immune-related and cell defense genes. Furthermore, real-time PCR analysis showed that levels of lysenin-related proteins mRNA in coelomic cells of <i>E. andrei</i> were upregulated after the injection of <i>Bacillus subtilis</i> bacteria. This EST data-set would provide a valuable resource for future researches of earthworm immune system.</p></div

Additional file 3: of Developmental biology and potential use of Alboglossiphonia lata (Annelida: Hirudinea) as an “Evo-Devo” model organism

Comparative Timeline of three glossiphoniidae leech species (Alboglossiphonia lata, Helobdella robusta and Helobdella austinensis) development from egg deposition (stage 1 at 0 h after zygote deposition (AZD)) until yolk-depleted juvenile. The embryonic development of A. lata is shorter than the one of both H. robusta and H. austinensis, with an approximate duration of seven and a half days (~180 h), compared to the approximated 9 and a half days (~229 h) for H. robusta and 13 days (~310 h) for H. austinensis. In A. lata, the vitelline membrane protecting the developing embryo is only broken during early stage 11 after the embryo has developed its eye spots and inverted its proboscis. In both H. robusta and H. austinensis, the vitelline membrane is broken by the maturing embryo somewhere between late stage 9 and early 10. (PPTX 81 kb

Additional file 1: of Developmental biology and potential use of Alboglossiphonia lata (Annelida: Hirudinea) as an “Evo-Devo” model organism

Phylogenetic tree of Ala-Calsensin. The evolutionary history was inferred using the Neighbor-Joining method [41]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [42]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [43] and are in the units of the number of amino acid substitutions per site. The analysis involved 8 amino acid sequences for Alboglossiphonia lata (Ala-calsensin), Helobdella robusta (Hro-calsensin protein id: 185,720), Haemopsis marmorata (Hma-calsensin protein id: AAC46630.1), Xenopus laevis (Xla-plastin3 protein id: NP_001083581.1), Cricetulus griseus (Cgr-plastin3 protein id: ERE65879), Anoplophora glabripennis (Agl-calbindin protein id: JAB67778), Drosophila melanogaster (Dme-calbindin32 protein id: AAA15214.1), Hydra vulgaris (Hvu-calbindin protein id: CDG71500). All ambiguous positions were removed from each sequence pair. There were a total of 318 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [44]. (PPTX 62 kb

Combined EGCG/As treatment increases apoptosis in BAEC.

<p>(A) BAEC were treated with various doses (0, 5, 10, 20, 30, or 40 μM) of As or EGCG for 24 h. (B) In some experiments, cells were also treated for 24 h with 20 μM EGCG, 20 μM As, or the combination of 20 μM EGCG and As each (EGCG/As). (A, B) Cell viability was measured using MTT assay. (C, D) Cells treated with EGCG, As, or EGCG/As for 12 h. (C) Apoptotic cells were detected by DAPI staining. (D) Cells were lysed in RIPA buffer. An equal amount (20 μg) of each cell lysate was subjected to Western blot analysis. Levels of cleaved PARP expression were detected with an anti-cleaved PARP antibody. Quantifications were performed using densitometry (Image J software) and results were normalized to β-actin. (E-G) The activity of caspases (3, 8, and 9) was measured in cells treated with EGCG, As or EGCG/As for the specified times (0, 6, 12, 18, or 24 h). All line graphs represent the relative caspase activity of the control. (H) Assay for Bax translocation into the mitochondria. Cells treated with EGCG, As, or EGCG/As for 12 h were stained with FITC-conjugated anti-Bax antibody, Mitotracker as a marker of mitochondria, or DAPI. All bar graphs represent the mean ± S.D. of 3 independent experiments. The different characters refer to significant differences (<i>P <</i> 0.05) among groups, which were determined by one-way ANOVA followed by post hoc Student-Newman-Keuls analysis.</p

NAC reverses cytotoxicity and pro-caspase activity induced by combined EGCG/As treatment.

<p>(A) BAEC were pretreated with various doses (0, 1, 5, or 10 mM) of NAC for 3 h prior to EGCG/As treatment for 24 h. (B-D) In separate experiments, EC were pretreated with the indicated dose (5 mM) of NAC. (E) In flow cytometric analysis, BAEC were pretreated with 20 μM Boc-D-FMK for 3 h prior to EGCG/As treatment for 12 h. (A) Cell viability, (B) caspase activity, (C) DAPI staining, (D) Bax translocation into the mitochondria, and (E) flow cytometric analyses were performed as described in the legend of Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g002" target="_blank">2</a>. All bar graphs represent the mean ± S.D. of 3 independent experiments. Statistical analysis was accomplished as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">Fig 1</a>.</p

Combined EGCG/As treatment increases ROS generation and decreases the activity of catalase but not SOD.

<p>(A) ROS levels were determined by flow cytometric analysis using DCFH-DA staining. Cells were treated with EGCG, As, or EGCG/As (each 20 μM) for 3 h and then stained with DCFH-DA. Stained cells were analyzed by flow cytometry using Cellquest software. The data are representative of 3 independent experiments. (B) SOD activity was assessed in cells treated with EGCG, As, or EGCG/As (each 20 μM) for 30 min. (C) The catalase activity was measured in EC treated with EGCG, As, or EGCG/As (each 20 μM) for 2.5 h. (D) Lipid peroxidation was estimated by measuring the production of malondialdehyde (MDA) using the Colorimetric Microplate Assay for Lipid Peroxidation Kit (Oxford) according to the manufacturer’s protocol. All bar graphs represent the mean ± S.D. of 3 independent experiments. Statistical analysis was accomplished as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">Fig 1</a>.</p

Combined EGCG/As decreases the viability of two types of EC, HUVEC and HBMEC.

<p>(A) HUVEC were prepared and treated with various doses (0, 10, 20, 30, 40, 50 or 100 μM) of As or EGCG for 24 h. (B) In separate experiments, HUVEC were also treated with 50 μM EGCG, 10 μM As, or the combination of 50 μM EGCG and 10 μM As (EGCG/As) for 24 h. In some experiments, cells were pretreated with 5 mM NAC, 50 U/ml catalase or 1 μM SP600125 for 30 min prior to exposed to EGCG/As. (C) HBMEC were prepared and treated with various doses (0, 10, 20, 30, 40, 50 or 100 μM) of As or EGCG for 24 h. (D) In separate experiments, cells were also treated with 50 M EGCG, 50 μM As, or the combination of 50 μM EGCG and As each (EGCG/As) for 24 h. In separate experiments, cells were pretreated with 5 mM NAC, 50 U/ml catalase or 10 μM SP600125 for 30 min prior to exposed to EGCG/As. Cell viability was measured using MTT assay. All bar graphs represent the mean ± S.D. of 3–5 independent experiments. Statistical analysis was accomplished as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">Fig 1</a>.</p

JNK mediates catalase activity, ROS production, and apoptosis altered by combined EGCG/As treatment.

<p>(A) BAEC were treated with EGCG/As for the indicated times (0, 0.5, 1, 2, or 3 h). (B) After pretreatment with catalase (50 U/ml) or the JNK inhibitor SP600125 (1 μM) for 30 min, EC were treated with EGCG/As for 1 h. The level of phosphorylated JNK (p-JNK) and total JNK protein was detected by Western blot analysis. (C) Cells were prepared and stained as described in the legends of Fig 5A and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g002" target="_blank">Fig 2</a>. In some experiments, cells were pretreated as described in the legend of Fig 5B, followed by treatment with EGCG/As for 3 h. Flow cytometric analysis was performed as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g002" target="_blank">Fig 2</a>. (D) Cell viability was determined as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">Fig 1</a> using BAEC pretreated with SP600125 prior to EGCG/As treatment for 24 h. (E) Catalase activity was measured as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g002" target="_blank">Fig 2</a> using BAEC pretreated with SP600125 prior to EGCG/As treatment for 2.5 h. (F, G) Cells were prepared, and pretreated with SP600125 (F) or MG132 (20 μM) (G) for 30 min prior to treatment of EGCG/As for 2.5 h. Cell lysate (30 μg) was subjected on 10% SDS-PAGE, and the level of catalase protein was then detected as described in <b>Materials and methods</b>. All bar graphs represent the mean±S.D. of 3 independent experiments. Statistical analysis was accomplished as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">Fig 1</a>.</p

Catalase reverses cytotoxicity and pro-caspase activity induced by combined EGCG/As treatment.

<p>BAEC pretreated with catalase (50 U/ml) for 30 min were exposed to EGCG/As for 24 h. (A) Cell viability, (B) Western blot analysis using the indicated antibodies, and (C) Bax translocation into the mitochondria were determined as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">Fig 1</a>. All bar graphs represent the mean ± S.D. of 3 independent experiments. Statistical analysis was accomplished as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138590#pone.0138590.g001" target="_blank">Fig 1</a>.</p

Structural Changes and Thermal Stability of Charged LiNi_<i>x</i>Mn_<i>y</i>Co_<i>z</i>O₂ Cathode Materials Studied by Combined <i>In Situ</i> Time-Resolved XRD and Mass Spectroscopy

Thermal stability of charged LiNi_<i>x</i>Mn_<i>y</i>Co_<i>z</i>O₂ (NMC, with <i>x</i> + <i>y</i> + <i>z</i> = 1, <i>x</i>:<i>y</i>:<i>z</i> = 4:3:3 (NMC433), 5:3:2 (NMC532), 6:2:2 (NMC622), and 8:1:1 (NMC811)) cathode materials is systematically studied using combined <i>in situ</i> time-resolved X-ray diffraction and mass spectroscopy (TR-XRD/MS) techniques upon heating up to 600 °C. The TR-XRD/MS results indicate that the content of Ni, Co, and Mn significantly affects both the structural changes and the oxygen release features during heating: the more Ni and less Co and Mn, the lower the onset temperature of the phase transition (i.e., thermal decomposition) and the larger amount of oxygen release. Interestingly, the NMC532 seems to be the optimized composition to maintain a reasonably good thermal stability, comparable to the low-nickel-content materials (e.g., NMC333 and NMC433), while having a high capacity close to the high-nickel-content materials (e.g., NMC811 and NMC622). The origin of the thermal decomposition of NMC cathode materials was elucidated by the changes in the oxidation states of each transition metal (TM) cations (i.e., Ni, Co, and Mn) and their site preferences during thermal decomposition. It is revealed that Mn ions mainly occupy the 3<i>a</i> octahedral sites of a layered structure (<i>R</i>3̅<i>m</i>) but Co ions prefer to migrate to the 8<i>a</i> tetrahedral sites of a spinel structure (<i>Fd</i>3̅<i>m</i>) during the thermal decomposition. Such element-dependent cation migration plays a very important role in the thermal stability of NMC cathode materials. The reasonably good thermal stability and high capacity characteristics of the NMC532 composition is originated from the well-balanced ratio of nickel content to manganese and cobalt contents. This systematic study provides insight into the rational design of NMC-based cathode materials with a desired balance between thermal stability and high energy density