Aspirin inhibits stem cell proliferation during freshwater Dugesia japonica regeneration by STAT3/SOX2/OCT4 signaling pathway.

As a widely used drug in clinical practice, aspirin has a large number of residual drugs and metabolites discharged into the environment during the pharmaceutical process or after taking the drug. Aspirin content and its metabolite, salicylic acid, have been reported and detected in several river water samples and municipal wastewaters. However, little is known about the toxicity mechanisms of this drug in aquatic invertebrates. In this study, we examine the toxic effect and investigate the toxicity mechanism of aspirin in planarian, which own the excellent regeneration and sensitive toxicity detection ability. Planarian is treated with 0.7 mM aspirin for 6 h, 48 h, 3 d and 5 d, and the mRNA and protein expression levels of the stem cells markers, in parallel with the target genes of the signaling pathway are analyzed by RT-qPCR, whole-mount immunofluorescence, and Western blot. The results show that aspirin strongly inhibits stem cell proliferation and causes retarded blastemas growth in planarians. Furthermore, the mRNA and protein expression levels of stem cells markers and the target genes dramatically decrease after the aspirin treatment. Meanwhile, the expression level of apoptotic cells also shows a downward trend. Their significant and coincident downregulations after the aspirin treatment suggest that aspirin regulates planarian regeneration via STAT3/SOX2/OCT4 signaling pathway. Our work reveals the toxicological effect and the mechanism of aspirin to the planarian, and provides basic data for therapeutic applications of aspirin in regeneration and warns about the ecological damage of aspirin abuse.

Photokinesis and Djopsin gene expression analysis during the regeneration of planarian eyes.

Planarians provide the ideal model for studying eye development, with their simple eye structure and exceptionally rapid regeneration. Here, we observed the eye morphogenesis, photophobic behavior, spectral sensitivity and expression pattern of Djopsin in the freshwater planarian Dugesia japonica. The results showed that: (i) Djopsin encoding the putative protein belonged to the rhabdomeric opsins group and displayed high conservation during animal evolution; (ii) planarians displayed diverse photophobic response to different visible wavelengths and were more sensitive to light blue (495 nm) and yellow (635 nm); (iii) the morphogenesis and functional recovery of eyes were related to the expression pattern of Djopsin during head regeneration; and (iv) Djopsin gene plays a major role in functional recovery during eye regeneration and visual system maintenance in adult planarians.

Identification and expression analysis of a Spsb gene in planarian Dugesia japonica.

The SPSB family is comprised of four highly conserved proteins, each containing a C-terminal SOCS box motif and a central SPRY domain. Presently, Spsb genes have been found in mammals and in a few invertebrates, however, the specific functions of these genes are still unknown. In this study, we identified a Spsb gene from the planarian Dugesia japonica and termed it DjSpsb. The temporal and spatial expression patterns of DjSpsb were examined in both intact and regenerative animals, and expression levels were also quantified in response to various stressors. The results show that (1) DjSpsb is highly conserved in evolutionary history in metazoans and is at closer relationship to Spsb1, Spsb2 and Spsb4; (2) DjSpsb mRNA is mainly expressed in the head and also throughout head regeneration processes, particularly, its expression up-regulated observably on day 5 after amputation; (3) DjSpsb is also expressed in the testes and yolk glands; (4) DjSpsb expression is induced by high temperature and ethanol but inhibited by high doses of ionic liquids. The date suggests that the DjSpsb gene might be active in central nervous system (CNS) formation and functional recovery during head regeneration, and it is also involved in the development of germ cells and stress responses in the planarians.

A pumilio homolog in Polycelis sp.

Pumilio proteins (PUMs), members of the pumilio/fem-3 mRNA-binding factor (PUF) family, are eukaryote-specific RNA-binding proteins. We isolated a 2,048-basepair cDNA fragment of a pumilio homolog from the planarian flatworm Polycelis sp. This pumilio protein (PyPUM) contains a conserved pumilio homology domain (PUM-HD) consisting of eight repeats and two flanking half repeats. PyPUM shows high similarity to Dugesia japonica pumilio (DjPUM) from another planarian D. japonica, and their PUM-HD also shows high similarity to each other. Furthermore, our data showed that there is a flatworm-specific spacer between repeats 7 and 8. Phylogenetic analysis showed that PyPUM has a closer relationship to other PUM homologs from flatworms. These results provide a foundation for future functional studies of pumilio gene in Polycelis sp.

Eight genes expression patterns during visual system regeneration in Dugesia japonica.

Dugesia japonica has become the suitable model system for studying the visual system molecular developmental processes because of their simple structure and high regenerative capacity. To further dissect the molecular events of genetic network controlling the visual system regeneration in D. japonica, we investigated the morphogenesis of regenerating eyes under the stereomicroscope and the transcripts expression levels of eight genes involved in this process by quantitative real-time PCR. The eight genes were Djeya, Djsix-1, Eye53, DjotxA, Djpax6, Djopsin, Djnetrin and 1020HH. The results showed that each gene was of different expression pattern at distinct regeneration stage and these eight genes could be divided into three groups according to the expression levels at different time points and the morphogenesis during eye reconstruction: (1) the early expression group, including Djeya, Djsix-1, Eye53, and DjotxA, which expression levels were significant increase from 1 to 3days after amputation; (2) the medium-term expression group, only including one gene, Djpax6, which expression level reached the peak on day 5; and (3) the late expression genes, including Djopsin, Djnetrin and 1020HH, which gradually increase transcription with the eye regeneration. Our data suggested that eye reconstruction was the results of polygenic services and the genes in the same group might have similar role or function in symphony.

Expression analysis of Djsix-1 gene during regeneration of planarian eyespots.

Djsix-1 gene is one of the important eyespots-regulating genes in planarians. In this experiment, the expression of Djsix-1 and morphogenesis of eyespots during planarians eyespots regeneration were investigated. The planarians were subjected to two rounds of transverse amputation. Nineteen time points in the first round and ten ones in the second one during the regeneration of the planarians post-auricle fragments were selected. At different time points, the quantitative expression of Djsix-1 and morphogenesis of eyespots during eyespots regeneration were examined by real-time RT-PCR and paraffin sections. Results showed that the optimal growth temperature for planarians regeneration was 22°C in dark. At this temperature, Djsix-1 gene was first up-regulated at the 30th min after the first round of transverse amputation and its expression level increased at the 24-h time point. The expression level reached peak on day 4 and then began to decrease thereafter. From day 6 to day 9, the expression level was maintained in a relatively stable level. In the second round, Djsix-1 expression reached the peak on the 2nd day after amputation, and then began to decrease and maintained in a relatively stable level from the 4th to the 9th day. Morphological and histological examinations showed that eyespots were observed on the 4th day after amputation in the first round and on the 3rd day in the second round. These results indicated that corresponding relationship existed between eyespots morphogenesis and Djsix-1 quantitative expression. It was also suggested that Djsix-1 promoted neoblasts proliferation at the early stage of eyespots regeneration and regulated morphogenesis of eyespots at the later stage.

Evaluation of endogenous reference genes for analysis of gene expression with real-time RT-PCR during planarian regeneration.

It is important that endogenous reference genes for real-time RT-PCR be empirically evaluated for stability in different cell types, developmental stages, and/or sample treatment. To select the most stable endogenous reference genes during planarian regeneration, three housekeeping genes, 18S rRNA, ACTB and DjEF2, were identified and established expression levels by real-time RT-PCR. The data were analyzed by GeNorm and NormFinder software. Expression levels of the Djsix-1 gene were studied in parallel with ACTB and DjEF2 both or each and 18S rRNA as reference during regeneration. The results showed that ACTB was the most stable expressed reference gene in the planarian regeneration.

Molecular characteristics of segment B of seven very virulent infectious bursal disease viruses isolated in China.

Infectious bursal disease virus (IBDV) is a birnavirus that causes immunosuppressive disease in chickens. Segment B of IBDV encodes the RNA-dependent RNA polymerase VP1, which is involved in virulence. We sequenced and analyzed segment B from seven Chinese IBDV isolates, all belonging to very virulent IBDV (vvIBDV), and clustered into Branches II and III. Phylogenetic analysis suggested that segment B of the HLJ isolates in Branch II might have originated from an unidentified host, and HuB-1 might have originated in Europe. Eight aa (4V, 61I, 145T, 287A, 508K, 511S, 646S, and 687P) were conserved in Branches II and III, and may contain potential segment B virulence determinants. Five aa (146D, 242E, 390M, 562P, and 695R) were found only in Branch III, and may be origin characteristics. Moreover, 55T and 63A in the 5'-untranslated region (UTR), and 2786C in the 3'-UTR were conserved in vvIBDV and may function in UTR secondary structure.

Sequence analysis of the VP2 hypervariable region of eight very virulent infectious bursal disease virus isolates from the northeast of China.

Nucleotide sequences of the VP2 gene of eight infectious bursal disease viruses isolated from vaccinated chicken flocks in the northeast of China were determined. The sequence analysis showed that all of the isolates were also characterized by the vvIBDV conserved amino acid residues: 222A, 256I, 294I, and 299S. Four of them had one amino acid change (D-->N) at position 212 in VP2 major hydrophilic peak A, while two of the four isolates had another one (A-->V) at position 321 in major hydrophilic peak B. The other isolates were similar to the UK661 strain. Our findings demonstrated that the vvlBDV strains in the northeast of China could be diverse.