SoxB family genes delay regeneration and cause abnormal movement in Dugesia japonica.

SoxB subfamily is an important branch of Sox family and plays a key role in animal physiological process, but little is known about their function in planarian regeneration. This study aims to evaluate the function of DjSoxB family genes in intact and regenerating planarians Dugesia japonica. Here, we amplify the full-length cDNA of DjSoxB1 and DjSoxB2 in D. japonica by rapid amplification of the cDNA ends (RACE), detect the expression of DjSoxB family genes in planarian. The results show that DjSoxBs are expressed in parenchymal tissue and the hybridization signals partially disappear after irradiation indicates DjSoxB family genes are expressed in neoblasts. After the RNA interference (RNAi) of DjSoxB1, DjSoxB2 and DjSoxB3 separately, the numbers of proliferative cells are all reduced that causes planarians show slower growth of blastema in the early stage of regeneration, and nerves of planarians are affected that the movement speed of planarians decreases in varying degrees. Specially, planarians in the DjSoxB3 RNAi group show shrinkage and twisting. Overall, this study reveals that DjSoxB family genes play a role in cell proliferation during regeneration. They also play an important role in the maintenance of normal nerve function and nerve regeneration. These results provide directions for the functional study of SoxB family genes and provide an important foundation for planarian regeneration.

Ecotoxicological evaluation and regeneration impairment of planarians by dibutyl phthalate.

Commonly utilized as a plasticizer in the food and chemical sectors, Dibutyl phthalate (DBP) poses threats to the environment and human well-being as it seeps or moves into the surroundings. Nevertheless, research on the harmfulness of DBP to aquatic organisms is limited, and its impact on stem cells and tissue regeneration remains unidentified. Planarians, recognized for their robust regenerative capabilities and sensitivity to aquatic pollutants, are emerging animal models in toxicology. This study investigated the comprehensive toxicity effects of environmentally relevant levels of DBP on planarians. It revealed potential toxicity mechanisms through the use of immunofluorescence, chromatin dispersion assay, Western blot, quantitative real-time fluorescence quantitative PCR (qRT-PCR), chromatin behavioral and histological analyses, immunofluorescence, and terminal dUTP nickel-end labeling (TUNEL). Findings illustrated that DBP caused morphological and motor abnormalities, tissue damage, regenerative inhibition, and developmental neurotoxicity. Further research revealed increased apoptosis and suppressed stem cell proliferation and differentiation, disrupting a balance of cell proliferation and death, ultimately leading to morphological defects and functional abnormalities. This was attributed to oxidative stress and DNA damage caused by excessive release of reactive oxygen species (ROS). This exploration furnishes fresh perspectives on evaluating the toxicity peril posed by DBP in aquatic organisms.

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.

PBDEs disrupt homeostasis maintenance and regeneration of planarians due to DNA damage, proliferation and apoptosis anomaly.

Polybrominated diphenyl ethers (PBDEs) are widely used as brominated flame retardants in the manufacturing industry, belonging to persistent organic pollutants in the environment. Planarians are the freshwater worms, with strong regenerative ability and extreme sensitivity to environmental toxicants. This study aimed to evaluate the potential acute comprehensive effects of PBDE-47/-209 on freshwater planarians. Methods to detect the effects include: detection of oxidative stress, observation of morphology and histology, detection of DNA fragmentation, and detection of cell proliferation and apoptosis. In the PBDE-47 treatment group, planarians showed increased oxidative stress intensity, severe tissue damage, increased DNA fragmentation level, and increased cell proliferation and apoptosis. In the PBDE-209 treatment group, planarians showed decreased oxidative stress intensity, slight tissue damage, almost unchanged DNA fragmentation level and apoptosis, proliferation increased only on the first day after treatment. In conclusion, both PBDE-47 and PBDE-209 are dangerous environmental hazardous material that can disrupt planarians homeostasis, while the toxicity of PBDE-47 is sever than PBDE-209 that PBDE-47 can lead to the death of planarians.

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.

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.

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.

Expression and functional analysis of flotillins in Dugesia japonica.

FLOTILLIN-1 and FLOTILLIN-2 are membrane rafts associated proteins that have been implicated in insulin and growth factor signaling, endocytosis, cell migration, proliferation, differentiation, cytoskeleton remodeling and membrane trafficking. Furthermore, FLOTILLINs also play important roles in the progression of cancer and neurodegenerative diseases. In this study, the roles of flotillins are investigated in planarian Dugesia japonica. The results show that Djflotillin-1 and Djflotillin-2 play a key role in homeostasis maintenance and regeneration process by regulating the proliferation of the neoblast cells, they are not involved in the maintenance and regeneration of the central nervous system in planarians.

The regulation of rapamycin in planarian Dugesia japonica Ichikawa & Kawakatsu, 1964 regeneration according to TOR signaling pathway.

The freshwater planarian mostly lives in the upper reaches of springs and rivers. Generally, it is realized as a suitable warning indicator of environmental toxicants. The freshwater planarian Dugesia japonica has a powerful regenerative capability and can regenerate a new individual including a complete central nervous system in one week. Rapamycin is an inhibitor of mammalian TORC1 (target of rapamycin complex-1) and used in the treatment of some diseases like cancer, cardiovascular and neurological diseases. However, the roles of rapamycin in the regulation of planarian regeneration remain to be elucidated. In present study, freshwater planarians D. japonica were firstly treated with 1 µM rapamycin for 18 h exposures and the expression patterns of Djtor was analyzed by the whole-mount in situ hybridization (WISH). Our results indicated rapamycin could strongly inhibit Djtor expression in planarian D. japonica and cause asymmetric blastemas and neuronal defects in planarians. Furthermore, knockdown of Djtor gene in planarians using RNA interference resulted in the suppression of downstream autophagy genes. These findings suggested that rapamycin might regulate freshwater planarian regeneration via Djtor signaling pathway.

Djhsp90s are crucial regulators during planarian regeneration and tissue homeostasis.

Heat shock protein 90 family members (HSP90s), as molecular chaperones, have conserved roles in the physiological processes of eukaryotes regulating cytoprotection, increasing host resistance and so on. However, whether HSP90s affect regeneration in animals is unclear. Planarians are emerging models for studying regeneration in vivo. Here, the roles of three hsp90 genes from planarian Dugesia japonica are investigated by WISH and RNAi. The results show that: (1) Djhsp90s expressions are induced by heat and cold shock, tissue damage and ionic liquid; (2) Djhsp90s mRNA are mainly distributed each side of the body in intact worms as well as blastemas in regenerative worms; (3) the worms show head regression, lysis, the body curling and the regeneration arrest or even failure after Djhsp90s RNAi; (4) Djhsp90s are involved in autophagy and locomotion of the body. The research results suggest that Djhsp90s are not only conserved in cytoprotection, but also involved in homeostasis maintenance and regeneration process by regulating different pathways in 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.

ADORA3: A Key Player in the Pathogenesis of Intracranial Aneurysms and a Potential Diagnostic Biomarker.

BACKGROUND: The effects of genes on the development of intracranial aneurysms (IAs) remain to be elucidated, and reliable blood biomarkers for diagnosing IAs are yet to be established. This study aimed to identify genes associated with IAs pathogenesis and explore their diagnostic value by analyzing IAs datasets, conducting vascular smooth muscle cells (VSMC) experiments, and performing blood detection. METHODS: IAs datasets were collected and the differentially expressed genes were analyzed. The selected genes were validated in external datasets. Autophagy was induced in VSMC and the effect of selected genes was determined. The diagnostic value of selected gene on the IAs were explored using area under curve (AUC) analysis using IAs plasma samples. RESULTS: Analysis of 61 samples (32 controls and 29 IAs tissues) revealed a significant increase in expression of ADORA3 compared with normal tissues using empirical Bayes methods of "limma" package; this was further validated by two external datasets. Additionally, induction of autophagy in VSMC lead to upregulation of ADORA3. Conversely, silencing ADORA3 suppressed VSMC proliferation and autophagy. Furthermore, analysis of an IAs blood sample dataset and clinical plasma samples demonstrated increased ADORA3 expression in patients with IA compared with normal subjects. The diagnostic value of blood ADORA3 expression in IAs was moderate when analyzing clinical samples (AUC: 0.756). Combining ADORA3 with IL2RB or CCR7 further enhanced the diagnostic ability for IAs, with the AUC value over 0.83. CONCLUSIONS: High expression of ADORA3 is associated with IAs pathogenesis, likely through its promotion of VSMC autophagy. Furthermore, blood ADORA3 levels have the potential to serve as an auxiliary diagnostic biomarker for IAs.

Revealing Novel Genes Related to Parkinson's Disease Pathogenesis and Establishing an associated Model.

Parkinson's disease (PD) represents a multifaceted neurological disorder whose genetic underpinnings warrant comprehensive investigation. This study focuses on identifying genes integral to PD pathogenesis and evaluating their diagnostic potential. Initially, we screened for differentially expressed genes (DEGs) between PD and control brain tissues within a dataset comprising larger number of specimens. Subsequently, these DEGs were subjected to weighted gene co-expression network analysis (WGCNA) to discern relevant gene modules. Notably, the yellow module exhibited a significant correlation with PD pathogenesis. Hence, we conducted a detailed examination of the yellow module genes using a cytoscope-based approach to construct a protein-protein interaction (PPI) network, which facilitated the identification of central hub genes implicated in PD pathogenesis. Employing two machine learning techniques, including XGBoost and LASSO algorithms, along with logistic regression analysis, we refined our search to three pertinent hub genes: FOXO3, HIST2H2BE, and HDAC1, all of which demonstrated a substantial association with PD pathogenesis. To corroborate our findings, we analyzed two PD blood datasets and clinical plasma samples, confirming the elevated expression levels of these genes in PD patients. The association of the genes with PD, as reflected by the area under the curve (AUC) values for FOXO3, HIST2H2BE, and HDAC1, were moderate for each gene. Collectively, this research substantiates the heightened expression of FOXO3, HIST2H2BE, and HDAC1 in both PD brain and blood samples, underscoring their pivotal contribution to the pathogenesis of PD.

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.

High rodent abundance increases seed removal but decreases scatter-hoarding and seedling recruitment along an elevational gradient.

The distributions of small rodents in mountainous environments across different elevations can provide important information regarding the effects of climate change on the dispersal of plant species. However, few studies of oak forest ecosystems have compared the elevational patterns of sympatric rodent diversity, seed dispersal, seed bank, and seedling abundance. Thus, we tested the differences in the seed disperser composition and abundance, seed dispersal, seed bank abundance, and seedling recruitment for Quercus wutaishanica along 10 elevation levels in the Taihang Mountains, China. Our results provide strong evidence that complex asymmetric seed dispersal and seedling regeneration exist along an elevational gradient. The abundance of rodents had a significant negative correlation with the elevation and the seed removal rates peaked and then declined with increasing elevation. The seed removal rates were higher at middle and lower elevations than higher elevations but acorns were predated by 5 species of seed predators at middle and lower elevations, and thus, there was a lower likelihood of recruitment compared with those dropped beneath mother oaks at higher elevations. More importantly, the number of individual seeds in the seed bank and seedlings increased with the elevation, although dispersal services were reduced at sites lacking rodents. As conditional mutualists, the rodents could possibly act as antagonistic seed predators rather than mutualistic seed dispersers at low and middle elevations, thereby resulting in the asymmetric pattern of rodent and seedling abundance with increasing elevation to affect the community assembly and ecosystem functions on a large spatial scale.

Anti-inflammatory effect of miR-125a-5p on experimental optic neuritis by promoting the differentiation of Treg cells.

Methylprednisolone pulse treatment is currently used for optic neuritis. It can speed visual recovery, but does not improve the ultimate visual outcomes. Recent studies have reported that miR-125a-5p has immunomodulatory effects on autoimmune diseases. However, it remains unclear whether miR-125a-5p has effects on optic neuritis. In this study, we used adeno-associated virus to overexpress or silence miR-125a-5p in mice. We found that silencing miR-125a-5p increased the latency of visual evoked potential and aggravated inflammation of the optic nerve. Overexpression of miR-125a-5p suppressed inflammation of the optic nerve, protected retinal ganglion cells, and increased the percentage of Treg cells. Our findings show that miR-125a-5p exhibits anti-inflammatory effects through promoting the differentiation of Treg cells.

Djptpn11 is indispensable for planarian regeneration by affecting early wound response genes expression and the Wnt pathway.

Planarian is an ideal model system of studying regeneration. Stem cell system and positional control genes (PCGs) are two important factors for perfect regeneration of planarians and they combine to promote their regeneration. Even so, how wounds regulate proliferation and neoblast fate is still important areas to address. Ptpn11 (Protein tyrosine phosphatase non-receptor type 11), one of PTP (Protein tyrosine phosphatase) family members, plays an important role in cellular processes including cell survival, proliferation, differentiation and apoptosis. Nevertheless, the role of ptpn11 in the planarian regeneration has not been fully studied. In this study, we identify the Djptpn11 gene to observe its function in planarian regeneration. The results reveal that the regeneration is severely inhibited and cause the disorder homeostasis in planarians. Furthermore, the stem cells proliferation and differentiation decreases while the apoptosis increases following Djptpn11 RNAi. At the same time, Djptpn11 affects the expression levels of early wound response genes (Djegr2, Dj1-jun, Djrunt1, Djwnt1 and Djnotum). Djwnt1 and Djnotum are two key Wnt signaling pathway genes and Djptpn11 affects the expression levels of Djwnt1 and Djnotum in the early and late stages of planarian regeneration. In general, Djptpn11 is indispensable for the homeostasis and regeneration of planarian by affecting the stem cells, early wound response genes and the Wnt pathway.

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.

Integrative taxonomy unveils a new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from the southern portion of the Taihang Mountains in northern China, with the description of its complete mitogenome and an exploratory analysis of mitochondrial gene order as a taxonomic character.

A new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from northern China is described on the basis of an integrative approach, involving morphology, karyology, histology, molecular distance, molecular phylogeny, and mitochondrial gene order. Here, we present the complete mitogenome of the new species Dugesia constrictiva Chen & Dong, sp. nov. This new species is mainly characterized by the presence of the following features: asymmetrical openings of the oviducts; large, cuboidal copulatory bursa; vasa deferentia opening through the ventro-lateral wall of the seminal vesicle; laterally compressed seminal vesicle; ventrally displaced ejaculatory duct, opening at the blunt tip of the penis papilla; long duct intercalated between seminal vesicle and diaphragm; chromosome complement diploid, with 16 metacentric chromosomes; mitochondrial gene order as follows: cox1-E-nad6-nad5-S2-D-R-cox3-I-Q-K-atp6-V-nad1-W-cox2-P-nad3-A-nad2-M-H-F-rrnS-L1-Y-G-S1-rrnL-L2-T-atp8-C-N-cob-nad4l-nad4. In triclads, mitochondrial gene order is considerably conserved between freshwater planarians and land flatworms, whereas it is variable between marine planarians and both freshwater and land flatworms. The secondary structures of tRNAs are all equipped with 4 arms, excepting tRNA S1 and tRNA S2, which lack the D arm and have excessively enlarged loops. Numerous transpositions of tRNA are present between D. constrictiva and its congeners. Mitochondrial gene arrangements may form a new, additional tool for taxonomic studies. The phylogenetic tree based on analysis of the mitochondrial genome basically corroborates current classification of the higher taxa of planarian flatworms.