Toxicity assessment of microcystin-leucine arginine in planarian Dugesia japonica.

Microcystin-leucine arginine (MC-LR), a representative cyanobacterial toxin, poses an increasing and serious threat to aquatic ecosystems. Despite investigating its toxic effects in various organisms and cells, the toxicity to tissue regeneration and stem cells in vivo still needs to be explored. Planarians are ideal regeneration and toxicology research models and have profound implications in ecotoxicology evaluation. This study conducted a systemic toxicity evaluation of MC-LR, including morphological changes, growth, regeneration, and the underlying cellular and molecular changes after MC-LR exposure, which were investigated in planarians. The results showed that exposure to MC-LR led to time- and dose-dependent lethal morphological changes, tissue damage, degrowth, and delayed regeneration in planarians. Furthermore, MC-LR exposure disturbed the activities of antioxidants, including total superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, and total antioxidant capacity, leading to oxidative stress and DNA damage, and then reduced the number of dividing neoblasts and promoted apoptosis. The results demonstrated that oxidative stress and DNA damage induced by MC-LR exposure caused apoptosis. Excessive apoptosis and suppressed neoblast activity led to severe homeostasis imbalance. This study explores the underlying mechanism of MC-LR toxicity in planarians and provides a basis for the toxicity assessment of MC-LR to aquatic organisms and ecological risk evaluation.

Djhsp70s, especially Djhsp70c, play a key role in planarian regeneration and tissue homeostasis by regulating cell proliferation and apoptosis.

Heat shock protein 70 family (HSP70s) is one of the most conserved and important group of HSPs as molecular chaperones, which plays an important role in cytoprotection, anti-apoptosis and so on. However, the molecular mechanism of HSP70s in animal regeneration remains to be delineated. In this study, we investigate the roles of HSP70s in regeneration of planarian. The four genes, Djhsp70a, Djhsp70b, Djhsp70c, and Djhsp70d of the HSP70s, are selected from the transcriptome database, because of their high expression levels in planarians. We then study the biological roles of each gene by conducting various experimental techniques, including RNAi, RT-PCR, WISH, Whole-mount immunostaining and TUNEL. The results show: (1) External stressors, such as temperature, tissue damage and ionic liquid upregulate the expression of Djhsp70s significantly. (2) The gene expression of Djhsp70s in planarians exhibits dynamic patterns. According to the result of WISH, the Djhsp70s are mainly expressed in parenchymal tissues on both sides of the body as well as blastema. It is consistent with the data of qRT-PCR. (3) After RNA interference of Djhsp70s, the worms experience cephalic regression and lysis, body curling, stagnant regeneration and death. (4) Knockdown of Djhsp70s affect the cell proliferation and apoptosis. These results suggest that Djhsp70s are not only conserved in cytoprotection, but involved in homeostasis maintenance and regeneration process by regulating coordination of cell proliferation and apoptosis in planarians.

Autophagy-related DjAtg1-1 plays critical role in planarian regeneration by regulating proliferation and cell death.

Autophagy is an intracellular degradation process and plays key roles in energy recycle and homeostasis maintenance during planarian regeneration. Although planarians provide an ideal model organism for studying autophagy in vivo, the molecular mechanism of planarian autophagy is still unknown. Here, we identify three autophagy-related (Atg) gene 1 homologs from Dugesia japonica and study their roles in planarian regeneration. Both DjATG1-1 and DjATG1-2 proteins show homology to vertebrate unc-51 like autophagy activating kinase 1 (ULK1) and ULK2, DjATG1-3 shows homology to vertebrate ULK3. In contrast to the ubiquitously expressed DjAtg1-1 and DjAtg1-3, DjAtg1-2 is mainly expressed in the intestine branches and epidermis. All the three DjAtg1s can respond to planarian regeneration and starvation. Both DjAtg1-1 and DjAtg1-2 are expressed in the reproductive organs of the starved sexual worms. DjAtg1-1 or DjAtg1-3 RNAi leads to head lysis and death of starved planarians, accompanied by exhaustion of neoblasts. DjAtg1-1 RNAi causes autophagy and regeneration defects and decreases proliferation and cell death; both DjAtg1-2 and DjAtg1-3 RNAi cause no autophagy or regeneration defect but increase cell death during regeneration. Our findings uncover the roles of DjAtg1s in autophagy and regeneration of planarian and highlight the links between proliferation, cell death, and autophagy during regeneration.

Djmek is involved in planarian regeneration by regulation of cell proliferation and apoptosis.

Dugesia japonica, belonging to Platyhelminthes, plays an important role in the animal evolution and is well known for its extraordinary regenerative ability. Mitogen activated protein kinase (MAPK) pathway is an important cell signaling pathway that converts extracellular stimuli into a wide range of cellular responses. The MAP-extracellular signal-regulated kinase (MEK) is a main component of MAPK/ERK signaling, but there are few studies on mek gene in planarians. In this study, we observe the expression patterns of Djmek1 and Djmek2 in planarians, and find that both of the two genes are required for the planarian regeneration. At the same time, we also find that both Djmek1 and Djmek2 are involved in the planarian regeneration by regulation of cell proliferation and apoptosis. Together, our findings show that the functions of the two genes are similar and complementary, and they play an important role in the regeneration of planarians.

Screening of Potential Key Transcripts Involved in Planarian Regeneration and Analysis of Its Regeneration Patterns by PacBio Long-Read Sequencing.

Dugesia japonica is an excellent animal model for studying the regeneration mechanism due to its characteristics of rapid regeneration and easy breeding. PacBio sequencing was performed on the intact planarians (In) and regenerating planarians of 1 day (1d), 3 days (3d), and 5 days (5d) after amputation. The aim of this study is to deeply profile the transcriptome of D. japonica and to evaluate its regenerate changes. Using robust statistical analysis, we identified 5931, 5115, and 4669 transcripts differentially expressed between 1d and In, 3d and In, 5d and In, respectively. A total of 63 key transcripts were screened from these DETs. These key transcripts enhance the expression in different regenerate stages respectively to regulate specific processes including signal transduction, mitosis, protein synthesis, transport and degradation, apoptosis, neural development, and energy cycling. Finally, according to the biological processes involved in these potential key transcripts, we propose a hypothesis of head regeneration model about D. japonica. In addition, the weighted gene co-expression network analysis provides a new way to screen key transcripts from large amounts of data. Together, these analyses identify a number of potential key regulators controlling proliferation, differentiation, apoptosis, and signal transduction. What's more, this study provides a powerful data foundation for further research on planarians regeneration.

Leptin-Sensitive JAK2 Activation in the Regulation of Tau Phosphorylation in PC12 Cells.

BACKGROUND/AIMS: Alzheimer's disease (AD) is characterized by two major hallmarks: the deposition and accumulation of ß-amyloid (Aß) peptide and hyperphosphorylated tau in intracellular neurofibrillary tangles. Sets of evidence show that leptin reduces Aß production and tau phosphorylation. Herein, we investigated the signaling pathways activated by leptin, to extensively understand its mechanism. METHODS: Western blotting was employed to assess the protein abundance of p-tau and BAX, MTT assay to decipher the cells viability. RESULTS: Leptin decreased tau phosphorylation, an effect was dependent on the activation of JAK2. CONCLUSION: The data suggest that JAK2 is involved in AD-related pathways.

Primarily screening and analyzing ESTs differentially expressed in rats' primary liver cancer.

OBJECTIVE: To screen and analyze key express sequence tags (ESTs) which were differentially displayed in every period of SD rats' primary hepatic carcinoma and reveal the molecular mechanism of carcinogenesis. METHODS: Using diethylnitrosamine (DENA) as a cancerigenic agent, animal models with different phases of primary hepatic cancer were constructed in SD rats. Rats were respectively sacrificed at d 14, d 28, d 56, d 77, d 105 and d 112 after the rats received DENA by gavage, then the livers were harvested. One part of the livers was classified according to their pathological changes, while the other was reserved for molecular mechanism studies on hepatocarcinogenesis. The differentially expressed genes were isolated from both normal and morbid tissues by mRNA differential display technique (DDRT-PCR). After the fragments were sequenced, bioinformatics were used to analyze the results. RESULTS: Twelve differentially expressed cDNA fragments were obtained. Nine fragments had the homology with known cDNA clones, especially EST-7 was similar to BN/SsNHsdMCW mitochondrion gene and the identity was 100% which suggested EST-7 may be the part of BN/SsNHsdMCW mitochondrion gene. In contrast, other three fragments (EST-1, EST-3 and EST-5) had extremely low identity to any genes registered in GENBANK databases. CONCLUSIONS: BN/SsNHsdMCW mitochondrion gene was expressed in different periods of hepatocarcinogenesis. Moreover, EST-1, EST-3 and EST-5 were suggested to contribute to the development of rat hepatocarcinogenesis, and thus may be candidates of new targets of oncogenes or cancer suppressor genes.

Changes of Serum Trace Elements, AFP, CEA, SF, T3, T4 and IGF-II in Different Periods of Rat Liver Cancer.

OBJECTIVE: Based on liver cancer model built in SD rats, the contents of trace elements (Cu, Fe, Zn, Ca and Mg), AFP, CEA, SF, TH and IGF-II in serum were measured at different stages to explore the molecular changes during the rat liver cancer development. METHODS: The SD rat liver cancer model was built by using diethylnitrosamine (DENA) as the mutagen. During 16 weeks of DENA gavage, blood samples were taken in the 14th, 28th, 56th, 77th, 105th and 112th days respectively after the first day of gavage with DENA, then the contents of five trace elements (Cu, Fe, Zn, Ca and Mg), T3, T4, IGF-II, AFP, CEA and SF in serum were determined. RESULTS: During the development of the rat liver cancer, in the test group, the Cu content significantly increased in serum, while the contents of Fe, Zn and Ca significantly decreased. The content of Mg showed no significant change. AFP and CEA of the test group showed same expression level with the control group; while the content of SF was lower than that of the control group when cancerization appeared. T3 and T4 increased at the first stage and then went down, and the content of IGF-II was always high. CONCLUSION: Cu, Fe, Zn, Ca, T3, T4, SF and IGF-II are closely related to the development of liver cancer. The changes of their contents in the development of cancer could enlighten the researches on cancer pathogenesis and prevention.