Exceptional Early Jurassic fossils with leathery eggs shed light on dinosaur reproductive biology.

Our understanding of pre-Cretaceous dinosaur reproduction is hindered by a scarcity of evidence within fossil records. Here we report three adult skeletons and five clutches of embryo-containing eggs of a new sauropodomorph from the Lower Jurassic of southwestern China, displaying several significant reproductive features that are either unknown or unlike other early-diverging sauropodomorphs, such as relatively large eggs with a relatively thick calcareous shell formed by prominent mammillary cones, synchronous hatching and a transitional prehatching posture between the crocodilians and living birds. Most significantly, these Early Jurassic fossils provide strong evidence for the earliest known leathery eggs. Our comprehensive quantitative analyses demonstrate that the first dinosaur eggs were probably leathery, elliptical and relatively small, but with relatively long eggshell units, and that along the line to living birds, the most significant change in reptilian egg morphology occurred early in theropod evolution rather than near the origin of Aves.

A new chronological framework for Chuandong Cave and its implications for the appearance of modern humans in southern China.

Chuandong Cave is an important Late Paleolithic site because it documents the early appearance of bone tools in southern China. We used the single-aliquot regenerative-dose protocol for optically stimulated luminescence dating to improve the precision of the chronology for the Chuandong Cave sedimentary sequence. The age of each layer was determined using a Bayesian modeling approach which combined optically stimulated luminescence ages with published AMS 14C dates. The results showed that Layer 10 began accumulating since 56 ± 14 ka and provides the upper age limit for all artifacts from the sequence. Bone awl tools from Layer 8, the earliest grinding bone tools in this site, were recovered within sediments between 40 ± 7 ka and 30 ± 4 ka. Layer 8 also indicates the appearance of modern humans in the Chuandong Cave sequence. Layers 4-2, ranging from 15 ± 3 ka until 11 ± 1 ka and including the Younger Dryas period, contain a few bone awls and an eyed bone needle. The shift from bone awls to eyed bone needles in the Chuandong Cave sequence indicates that modern humans adapted to the changing climate of southern China. We conclude that modern human behavior in bone tools appeared in southern China as early as 40 ± 7 ka, became more sophisticated during the Last Glacial Maximum, and spread more widely across southern China during the Younger Dryas.

N-acetylcysteine alleviated paraquat-induced mitochondrial fragmentation and autophagy in primary murine neural progenitor cells.

The developing brain is uniquely vulnerable to toxic chemical exposures. Studies indicate that neural stem cell (NSC) self-renewal is susceptible to oxidative stress caused by xenobiotics. However, the impact of antioxidants on NSC self-renewal and the potential mechanisms remain elusive. In this study, primary murine neural progenitor cells (mNPCs) from the subventricular zone were used as a research model. In addition, paraquat (PQ) was used to elicit oxidative stress and N-acetylcysteine (NAC) was used as a powerful antioxidant. mNPCs were treated with 80 µm PQ for 24 hours with or without 4 hours of NAC pretreatment. Our results showed that PQ treatment increased intracellular reactive oxygen species production, decreased cell viability and DNA synthesis, and promoted cell apoptosis. Meanwhile, pretreatment with NAC alleviated PQ-induced cytotoxicity in mNPCs. To elucidate the mechanisms further, we found that NAC pretreatment prevented PQ-induced reactive oxygen species production, mitochondrial fragmentation and autophagy in mNPCs. NAC-pretreated cells showed increased anti-apoptotic protein Bcl-2 and decreased pro-apoptotic protein Bax expression. Similarly, NAC pretreatment increased p-mTOR and decreased LC3B-II protein expression. Moreover, NAC decreased mitophagy related mRNA Pink1 and Parkin expression. Taken together, our results suggested that the antioxidant NAC treatment significantly attenuated PQ-induced mNPC self-renewal disruption through decreasing autophagy and salvaging mitochondrial morphology. These findings revealed a potential mechanism for neurological treatment relating to antioxidant and suggested potentially relevant implications for PQ-related neurodegenerative disorders. Thus, our study also provided insight into therapeutic strategies for the neurotoxic effects of oxidative stress-associated toxicants.

Modification of Wnt signaling pathway on paraquat-induced inhibition of neural progenitor cell proliferation.

Paraquat (PQ) is an agricultural chemical used worldwide. As a potential neurotoxicant, PQ adversely affects neurogenesis and inhibits proliferation of neural progenitor cells (NPCs). However, the molecular mechanistic insights of PQ exposure on NPCs remains to be determined. Herein, we determine the extent to which Wnt/ß-catenin signaling involved in the inhibition effect of PQ on mouse NPCs from subventricular zone (SVZ). NPCs were treated with different concentrations of PQ (40, 80, and 120 µM). PQ exposure provoked oxidative stress and apoptosis and PQ inhibited cell viability and proliferation in a concentration-dependent manner. Significantly, PQ exposure altered the expression/protein levels of the Wnt pathway genes in NPCs. In addition, PQ reduced cellular ß-catenin, p-GSK-3ß, and cyclin-D1 and increased the radio of Bax/Bcl2. Further, Wnt pathway activation by treatment with LiCl and Wnt1 attenuated PQ-induced inhibition of mNPCs proliferation. Antioxidant (NAC) treatment alleviated the inhibition of PQ-induced Wnt signaling pathway. Overall, our results suggest significant inhibitory effects of PQ on NPCs proliferation via the Wnt/ß-catenin signaling pathway. Interestingly, our results implied that activation of Wnt/ß-catenin signaling pathway attenuated PQ-induced autophagic cell death. Our results therefore bring our understanding of the molecular mechanisms of PQ-induced neurotoxicity.

Integrated analysis of paraquat-induced microRNAs-mRNAs changes in human neural progenitor cells.

Paraquat (PQ), one of the most widely used fast-acting and non-selective herbicides in the world is believed to act as a neurotoxicant, which increases the risk of developing Parkinson's disease (PD) by selectively impairing dopaminergic neurons. However, the mechanism of PQ on neural progenitor cells remains unclear. As regulator of mRNA expression, miRNA play a crucial role in neurotoxicity. Based on our previous study, we chose 10µM PQ, which induced ROS production and inhibited proliferation but not reduced the cell viability. In this study, we present an integrative analysis of PQ-induced whole transcriptome changes and its regulatory miRNA networks in human neural progenitor cells (hNPCs). Integrated analysis of PQ-induced miRNA-mRNA alteration reveals differential expression of 3972 mRNAs and 52 miRNAs. Based on the GO analysis and pathway analysis of the intersection genes, we found 48 significantly altered GO terms and 22 significant pathways, among which, Wnt signaling being the top-ranked pathway. We verified that the expression of 9 miRNAs and 11 mRNAs related to the Wnt signaling pathway were altered in a dose-dependent manner by qPCR. These results indicate that PQ changes mRNAs and miRNAs expression in hNPCs, leading to the alteration of several neurodevelopment related key biological processes and crucial pathways, especially Wnt signaling pathway. Moreover, it suggests that PQ could downregulate Wnt signaling pathway via miRNA to induce developmental neurotoxicity.

Low-Dose Methylmercury-Induced Genes Regulate Mitochondrial Biogenesis via miR-25 in Immortalized Human Embryonic Neural Progenitor Cells.

Mitochondria are essential organelles and important targets for environmental pollutants. The detection of mitochondrial biogenesis and generation of reactive oxygen species (ROS) and p53 levels following low-dose methylmercury (MeHg) exposure could expand our understanding of underlying mechanisms. Here, the sensitivity of immortalized human neural progenitor cells (ihNPCs) upon exposure to MeHg was investigated. We found that MeHg altered cell viability and the number of 5-ethynyl-2'-deoxyuridine (EdU)-positive cells. We also observed that low-dose MeHg exposure increased the mRNA expression of cell cycle regulators. We observed that MeHg induced ROS production in a dose-dependent manner. In addition, mRNA levels of peroxisome-proliferator-activated receptor gammacoactivator-1α (PGC-1α), mitochondrial transcription factor A (TFAM) and p53-controlled ribonucleotide reductase (p53R2) were significantly elevated, which were correlated with the increase of mitochondrial DNA (mtDNA) copy number at a concentration as low as 10 nM. Moreover, we examined the expression of microRNAs (miRNAs) known as regulatory miRNAs of p53 (i.e., miR-30d, miR-1285, miR-25). We found that the expression of these miRNAs was significantly downregulated upon MeHg treatment. Furthermore, the overexpression of miR-25 resulted in significantly reducted p53 protein levels and decreased mRNA expression of genes involved in mitochondrial biogenesis regulation. Taken together, these results demonstrated that MeHg could induce developmental neurotoxicity in ihNPCs through altering mitochondrial functions and the expression of miRNA.

Low-Dose Methylmercury-Induced Apoptosis and Mitochondrial DNA Mutation in Human Embryonic Neural Progenitor Cells.

Methylmercury (MeHg) is a long-lasting organic pollutant primarily found in the aquatic environment. The developing brain is particularly sensitive to MeHg due to reduced proliferation of neural stem cell. Although several mechanisms of MeHg-induced apoptosis have been defined in culture models, it remains unclear whether mitochondrial DNA (mtDNA) mutation is involved in the toxic effect of MeHg, especially in the neural progenitor cells. In the present study, the ReNcell CX cell, a human neural progenitor cells (hNPCs) line, was exposed to nanomolar concentrations of MeHg (≤50 nM). We found that MeHg altered mitochondrial metabolic function and induced apoptosis. In addition, we observed that MeHg induced ROS production in a dose-dependent manner in hNPCs cells, which was associated with significantly increased expressions of ND1, Cytb, and ATP6. To elucidate the mechanism underlying MeHg toxicity on mitochondrial function, we examined the ATP content and mitochondrial membrane potential in MeHg-treated hNPCs. Our study showed that MeHg exposure led to decreased ATP content and reduced mitochondrial membrane potential, which failed to match the expansion in mtDNA copy number, suggesting impaired mtDNA. Collectively, these results demonstrated that MeHg induced toxicity in hNPCs through altering mitochondrial function and inducing oxidative damage to mtDNA.

Nrf2/ARE Pathway Involved in Oxidative Stress Induced by Paraquat in Human Neural Progenitor Cells.

Compelling evidences have shown that diverse environmental insults arising during early life can either directly lead to a reduction in the number of dopaminergic neurons or cause an increased susceptibility to neurons degeneration with subsequent environmental insults or with aging alone. Oxidative stress is considered the main effect of neurotoxins exposure. In this study, we investigated the oxidative stress effect of Paraquat (PQ) on immortalized human embryonic neural progenitor cells by treating them with various concentrations of PQ. We show that PQ can decrease the activity of SOD and CAT but increase MDA and LDH level. Furthermore, the activities of Cyc and caspase-9 were found increased significantly at 10 µM of PQ treatment. The cytoplasmic Nrf2 protein expressions were upregulated at 10 µM but fell back at 100 µM. The nuclear Nrf2 protein expressions were upregulated as well as the downstream mRNA expressions of HO-1 and NQO1 in a dose-dependent manner. In addition, the proteins expression of PKC and CKII was also increased significantly even at 1 µM. The results suggested that Nrf2/ARE pathway is involved in mild to moderate PQ-induced oxidative stress which is evident from dampened Nrf2 activity and low expression of antioxidant genes in PQ induced oxidative damage.

[The effect of Wnt signaling pathway on paraquat induced PC12 cells damage].

OBJECTIVE: To investigate the role of Wnt signaling pathway on paraquat (PQ)induced PC12 cells damage. METHODS: Using PC12 cells, in this study CCK8 assay was used to detect the effect of cell viability. The cell apoptosis and cell cycle was detected by flow cytometry. The real-time polymerase chain reaction (RT-PCR) was used to measure the mRNA expression of Wnt pathway key genes including Fzd1, Dvl2 and ß-catenin and downstream genes including Bax, Bcl2, Survivin, Cyclin D1 and C-myc. RESULT: Compared with the control, PC12 cells viability in 50.00 and 100.00 µmol/L PQ treatment groups were obviously decreased, the cell cycle S phase arrest, and cell apoptosis increased (P<0.05). The 25.00, 50.00 and 100.00 µmol/L PQ treatment groups mRNA expression of Wnt pathway key genes including Fzd1, Dvl2 and ß-catenin and downstream genes including apoptosis suppressor genes (Bcl-2 and survivin)and cyclin gene (Cyclin D1) were downregulated (P<0.05). The mRNA expression of pro-apoptosis gene (Bax) and cyclin gene (C-myc) were upregulated (P<0.05). CONCLUSION: It suggested that PQ can activate Wnt pathway to regulate downsteam genes expression, resulting in PC12 cell cycle arrest and apoptosis.

Characterization of paraquat-induced miRNA profiling response in hNPCs undergoing proliferation.

Aberration during the development of the central nervous system (CNS) due to environmental factors underlies a variety of adverse developmental outcomes. Paraquat (PQ) is a widely studied neurotoxicant that perturbs the normal structure/function of adult CNS. Yet, the impacts of PQ exposure on the developing CNS remain unclear. miRNAs represent a class of small non-coding RNA molecules involved in the regulation of neural development. Thus in the present study, we analyzed the impacts of PQ on the miRNome of human neural progenitor cells (hNPCs) during proliferation by using the Exiqon miRCURY™ LNA Array. A total of 66 miRNAs were identified as differentially expressed in proliferating hNPCs upon PQ treatment. miRTarBase prediction identified 1465 mRNAs, including several genes (e.g., nestin, sox1, ngn1) previously proved to be associated with the neural proliferation and differentiation, as target genes of PQ-induced differentially expressed miRNAs. The database for annotation, visualization and integrated discovery (DAVID) bioinformatics analysis showed that target genes were enriched in regulation of cell proliferation and differentiation, cell cycle and apoptosis as well as tumor protein 53 (p53), Wnt, Notch and mitogen-activated protein kinases (MAPK) signaling pathways (p < 0.001). These findings were confirmed by real-time RT-PCR. Based on our results we conclude that PQ-induced impacts on the miRNA profiling of hNPCs undergoing proliferation may underlie the developmental neurotoxicity of PQ.

Paraquat inhibits cell viability via enhanced oxidative stress and apoptosis in human neural progenitor cells.

Paraquat (PQ) is one of the most widely used herbicides in the world. Although available evidence indicates that people exposed to PQ have a higher risk of developing Parkinson's disease, adverse effects of PQ on neural progenitor cells have not been investigated yet. In this study, we investigated the in vitro effect of PQ on immortalized human embryonic neural progenitor cells (hNPCs) by treating them with various concentrations of PQ (0, 0.1, 1, 10 and 100 µM) for 24h. We show that PQ treatment reduces the cell viability and proliferation and induces reactive oxygen species (ROS) production in a dose-dependent manner. In addition, apoptosis induced by PQ was significantly increased at a concentration of as low as 1 µM. To illustrate the underlying molecular mechanisms, we examined the caspase-3 activity, intracellular calcium level, the NF-κB activity, as well as expression of p21, p53 and metallothionein-III mRNA. PQ significantly increased caspase-3 activity at the concentration of 100 µM. Similarly, PQ triggered intracellular Ca(2+) releases and activation of NF-κB was observed after exposure of hNPCs at low concentrations of PQ (1 µM). Meanwhile, p53 and p21 mRNA transcripts were significantly up-regulated at 10 µM and 1 µM of PQ, respectively. MT-III mRNA and protein expression was significantly up-regulated at 1 µM of PQ and reached peak at 10 µM. These results suggest that PQ could reduce viability of hNPCs by inducing oxidative stress and apoptosis.