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.

[Effects of ZnO nanoparticles on proliferation and apoptosis of human lung epithelial BEAS-2B cells].

OBJECTIVE: To investigate the effects of ZnO nanoparticles (ZnO NPs) on proliferation and apoptosis of human lung epithelial cells BEAS-2B and its molecular mechanisms. METHODS: BEAS-2B cells were treated with ZnO NPs at concentrations of 3, 6 and 12 µg/ml for 12 h and 24 h, the control group was not treated with ZnO NPs, each with 3 replicate wells. Cell viability was detected by CCK-8 method, and the half lethal concentration (IC50) was analyzed. Then, the BEAS-2B cells were treated with ZnO NPs at selected concentrations of 3 and 6 µg/ml for 24 h respectively,each group was set with 3 replicate. Cell morphology was observed under inverted microscope. The morphology of cell nuclei was observed by Hochest33342 staining. The morphology of apoptosis was observed by AO staining and scanning electron microscopy. Cell cycle progression, cell apoptosis rate and the level of reactive oxygen species(ROS)were detected by flow cytometry. Western blot was used to detect the expression levels of Bcl-2 and Bax protein. RESULTS: Compared with the control group, the cell viability of cells treated with ZnO NPs were decreased significantly(P＜0.01), and the IC50 was 6.13 µg/ml at 24 h of drug treatment. After the cells were treated with ZnO NPs for 24 h, the levels of ROS were increased significantly(P＜0.05, P＜0.01)in 3 µg/ml, 6 µg/ml groups. The cell cycle was arrested at G2/M phase, chromatin condensation and apoptotic bodies were induced, apoptosis rate was increased significantly(P＜0.01) in 6 µg/ml group. The expression of Bcl-2 was decreased(P＜0.05), and the expression of Bax was increased (P＜0.05) in cells treated with 6 µg/ml ZnO NPs for 24 h. CONCLUSION: ZnO NPs induced ROS accumulation, blocked progress of cell cycle and induced cell apoptosis in BEAS-2B cells.

Two new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from the tropical monsoon forest in southern China.

Two new species of the genus Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from the tropical monsoon forest in southern China are described on the basis of an integrative taxonomic study involving morphology, karyology, histology, and molecular analyses. The new species Dugesiacircumcisa Chen & Dong, sp. nov. is characterised by asymmetrical openings of the oviducts; right vas deferens opening at anterior portion of the seminal vesicle and the left one opening at mid-lateral portion of the seminal vesicle; two diaphragms in ejaculatory duct, the latter being ventrally displaced and opening at the tip of the penis papilla, which is provided with a nozzle; wide duct connecting male atrium and common atrium; chromosome complement triploid with 24 metacentric chromosomes. The other new species, Dugesiaverrucula Chen & Dong, sp. nov., is characterised by the large size of the living worm, usually exceeding 3.5 cm in length; asymmetrical openings of the oviducts; subterminal opening of ventrally displaced ejaculatory duct; vasa deferentia symmetrically opening into the postero-lateral portion of the seminal vesicle; well-developed duct between the seminal vesicle and diaphragm; single dorsal bump near the root of the penis papilla; bursal canal with pleated wall and spacious posterior section; unstalked cocoons; chromosome complement diploid with 16 metacentric chromosomes. Inter-specific molecular distances and their positions in the phylogenetic tree reveal that D.circumcisa and D.verrucula are clearly separated from their congeners.

The negative effect of the PI3K inhibitor 3-methyladenine on planarian regeneration via the autophagy signalling pathway.

As an important PI3K (VPS34) inhibitor, 3-methyladenine (3-MA) can block the formation of autophagic vesicles in animals. Most toxicological studies using 3-MA have shown that 3-MA leads to serious disorders via autophagy suppression in mammals. However, no toxicological research on 3-MA has been performed on individuals undergoing regeneration. The freshwater planarian has powerful regenerative capability, and it can regenerate a new brain in 5 days and undergo complete adult individual remodelling in approximately 14 days. Moreover, it is also an excellent model organism for studies on environmental toxicology due to its high chemical sensitivity and extensive distribution. Here, Dugesia japonica planarians were treated with 3-MA, and the results showed that autophagy was inhibited and Djvps34 expression levels were down-regulated. After exposure to 10 mM 3-MA for 18 h, all the controls showed normal phenotypes, while one-half of the planarians treated with 3-MA showed morphological defects. In most cases, an ulcer appeared in the middle of the body, and a normal phenotype was restored 7 days following 3-MA exposure. During regeneration, disproportionate blastemas with tissue regression were observed. Furthermore, 3-MA treatment suppressed stem cell proliferation in intact and regenerating worms. These findings demonstrate that autophagy is indispensable for tissue homeostasis and regeneration in planarians and that 3-MA treatment is detrimental to planarian regeneration via its effect on the autophagy pathway.

Gamma-Secretase Inhibitor (DAPT), a potential therapeutic target drug, caused neurotoxicity in planarian regeneration by inhibiting Notch signaling pathway.

DAPT (N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester) is a γ-secretase inhibitor that indirectly blocks the activity of Notch pathway. It is a potential therapeutic target drug for many diseases, such as cancer, neurological, cardiovascular, and cerebrovascular diseases. However, the pharmacological action and specific mechanisms of DAPT are not clear. Planarians have strong regenerative capacity and can regenerate a new individual with a complete nervous system in one week. Thus, they are used as an ideal indicator of environmental toxicants and a novel model for studying neurodevelopmental toxicology. In this study, different concentrations and treatment times of DAPT are used to analyze the gene expression levels of major components in Notch pathway. The results show that the optimal concentration and exposure time of DAPT is 100 nM for 10 days in planarians and indicate that the inhibitory of DAPT treatment on Notch pathway is time- and concentration-dependent. The potential impact of DAPT is effectively analyzed by qPCR, WISH, and Immunofluorescence. The results indicate that DAPT exposure causes intact planarian wavy or swollen, and regenerative planarians asymmetric growth or muti-eye. Moreover, DAPT exposure increases cell proliferation and apoptosis, results in neurodevelopmental defects and dynamic changes of some marker genes. These results suggest that the balance of proliferation and apoptosis is disturbed, and then, affecting tissue homeostasis and differentiation. These findings demonstrate that DAPT has serious side effects in organisms and relies on Notch pathway to determine cell fate, it is cautious in the use of DAPT as a potential therapeutic approach for the disease in clinical trials.

Antioxidant responses and lipid peroxidation can be used as sensitive indicators for the heavy metals risk assessment of the Wei River: a case study of planarian Dugesia Japonica.

PURPOSE: To verify antioxidant responses and lipid peroxidation can be used as sensitive indicators for the risk assessment of the Wei River. MATERIAL AND METHODS: We investigate the effects of the Wei River on oxidative stress of planarian Dugesia japonica by antioxidant parameters, and use ICP-MS to measure the heavy metals in the Wei River. Then, we observe the effects of three common heavy metal ions (Cr3+, Hg2+, Pb2+) on the regeneration of planarians on morphological and histological levels. RESULTS: The significant changes of antioxidant parameters (SOD, CAT, GPx, GST, T-AOC) and MDA content indicate that oxidative stress is induced after the Wei River exposure on planarians, though the heavy metals in the Wei River are not exceeding the standards. Then, the regeneration of planarians shows different degree of morphological and histological damage after Cr3+, Hg2+ and Pb2+ exposure. CONCLUSION: We speculate that the heavy metal ions in the Wei River, especially Cr3+, Hg2+ and Pb2+, may give rise to oxidative damage on planarians. These findings illustrate that planarian can serve as an indicator of aquatic ecosystem pollution, antioxidant responses and lipid peroxidation can also be used as sensitive indicators and provide an excellent opportunity for urban river risk assessment.

Roles of mTOR Signaling in Tissue Regeneration.

The mammalian target of rapamycin (mTOR), is a serine/threonine protein kinase and belongs to the phosphatidylinositol 3-kinase (PI3K)-related kinase (PIKK) family. mTOR interacts with other subunits to form two distinct complexes, mTORC1 and mTORC2. mTORC1 coordinates cell growth and metabolism in response to environmental input, including growth factors, amino acid, energy and stress. mTORC2 mainly controls cell survival and migration through phosphorylating glucocorticoid-regulated kinase (SGK), protein kinase B (Akt), and protein kinase C (PKC) kinase families. The dysregulation of mTOR is involved in human diseases including cancer, cardiovascular diseases, neurodegenerative diseases, and epilepsy. Tissue damage caused by trauma, diseases or aging disrupt the tissue functions. Tissue regeneration after injuries is of significance for recovering the tissue homeostasis and functions. Mammals have very limited regenerative capacity in multiple tissues and organs, such as the heart and central nervous system (CNS). Thereby, understanding the mechanisms underlying tissue regeneration is crucial for tissue repair and regenerative medicine. mTOR is activated in multiple tissue injuries. In this review, we summarize the roles of mTOR signaling in tissue regeneration such as neurons, muscles, the liver and the intestine.

Single-cell analyses identify distinct and intermediate states of zebrafish pancreatic islet development.

Pancreatic endocrine islets are vital for glucose homeostasis. However, the islet developmental trajectory and its regulatory network are not well understood. To define the features of these specification and differentiation processes, we isolated individual islet cells from TgBAC(neurod1:EGFP) transgenic zebrafish and analyzed islet developmental dynamics across four different embryonic stages using a single-cell RNA-seq strategy. We identified proliferative endocrine progenitors, which could be further categorized by different cell cycle phases with the G1/S subpopulation displaying a distinct differentiation potential. We identified endocrine precursors, a heterogeneous intermediate-state population consisting of lineage-primed alpha, beta and delta cells that were characterized by the expression of lineage-specific transcription factors and relatively low expression of terminally differentiation markers. The terminally differentiated alpha, beta, and delta cells displayed stage-dependent differentiation states, which were related to their functional maturation. Our data unveiled distinct states, events and molecular features during the islet developmental transition, and provided resources to comprehensively understand the lineage hierarchy of islet development at the single-cell level.

Ribosome biogenesis protein Urb1 acts downstream of mTOR complex 1 to modulate digestive organ development in zebrafish.

Ribosome biogenesis is essential for the cell growth and division. Disruptions in ribosome biogenesis result in developmental defects and a group of diseases, known as ribosomopathies. Here, we report a mutation in zebrafish urb1, which encodes an essential ribosome biogenesis protein. The urb1cq31 mutant exhibits hypoplastic digestive organs, which is caused by impaired cell proliferation with the differentiation of digestive organ progenitors unaffected. Knockdown of mtor or raptor leads to similar hypoplastic phenotypes and reduced expression of urb1 in the digestive organs. Overexpression of Urb1 results in overgrowth of digestive organs, and can efficiently rescue the hypoplastic liver and pancreas in the mtor and raptor morphants. Reduced syntheses of free ribosomal subunits and impaired assembly of polysomes are observed in the urb1 mutant as well as in the mtor and raptor morphants, which can be rescued by the Urb1 overexpression. These data demonstrate that Urb1 plays an important role in governing ribosome biogenesis and protein synthesis downstream of mammalian/mechanistic target of rapamycin complex 1 (mTORC1), thus regulating the development of digestive organs. Our study indicates the requirement of hyperactive protein synthesis for the digestive organ development.