Structure-based prediction and characterization of photo-crosslinking in native protein-RNA complexes.

UV-crosslinking of protein and RNA in direct contacts has been widely used to study protein-RNA complexes while our understanding of the photo-crosslinking mechanisms remains poor. This knowledge gap is due to the challenge of precisely mapping the crosslink sites in protein and RNA simultaneously in their native sequence and structural contexts. Here we systematically analyze protein-RNA interactions and photo-crosslinking by bridging crosslinked nucleotides and amino acids mapped using different assays with protein-RNA complex structures. We developed a computational method PxR3D-map which reliably predicts crosslink sites using structural information characterizing protein-RNA interaction interfaces. Analysis of the informative features revealed that photo-crosslinking is facilitated by base stacking with not only aromatic residues, but also dipeptide bonds that involve glycine, and distinct mechanisms are utilized by different RNA-binding domains. Our work suggests protein-RNA photo-crosslinking is highly selective in the cellular environment, which can guide data interpretation and further technology development for UV-crosslinking-based assays.

Automated vertebral bone mineral density measurement with phantomless internal calibration in chest LDCT scans using deep learning.

OBJECTIVE: To develop and evaluate a fully automated method based on deep learning and phantomless internal calibration for bone mineral density (BMD) measurement and opportunistic low BMD (osteopenia and osteoporosis) screening using chest low-dose CT (LDCT) scans. METHODS: A total of 1175 individuals were enrolled in this study, who underwent both chest LDCT and BMD examinations with quantitative computed tomography (QCT), by two different CT scanners (Siemens and GE). Two convolutional neural network (CNN) models were employed for vertebral body segmentation and labeling, respectively. A histogram technique was applied for vertebral BMD calculation using paraspinal muscle and surrounding fat as references. 195 cases (by Siemens scanner) as fitting cohort were used to build the calibration function. 698 cases as validation cohort I (VCI, by Siemens scanner) and 282 cases as validation cohort II (VCII, by GE scanner) were performed to evaluate the performance of the proposed method, with QCT as the standard for analysis. RESULTS: The average BMDs from the proposed method were strongly correlated with QCT (in VCI: r = 0.896, in VCII: r = 0.956, p < 0.001). Bland-Altman analysis showed a small mean difference of 1.1 mg/cm3, and large interindividual differences as seen by wide 95% limits of agreement (-29.9 to +32.0 mg/cm3) in VCI. The proposed method measured BMDs were higher than QCT measured BMDs in VCII (mean difference = 15.3 mg/cm3, p < 0.001). Osteoporosis and low BMD were diagnosed by proposed method with AUCs of 0.876 and 0.903 in VCI, 0.731 and 0.794 in VCII, respectively. The AUCs of the proposed method were increased to over 0.920 in both VCI and VCII after adjusting the cut-off. CONCLUSION: Without manual selection of the region of interest of body tissues, the proposed method based on deep learning and phantomless internal calibration has the potential for preliminary screening of patients with low BMD using chest LDCT scans. However, the agreement between the proposed method and QCT is insufficient to allow them to be used interchangeably in BMD measurement. ADVANCES IN KNOWLEDGE: This study proposed an automated vertebral BMD measurement method based on deep learning and phantomless internal calibration with paraspinal muscle and fat as reference.

Tumor cells-derived exosomal PD-L1 promotes the growth and invasion of lung cancer cells in vitro via mediating macrophages M2 polarization.

Lung cancer originating from the bronchial epithelium is the most common lung malignancy. It has been reported that programmed cell death 1 ligand 1 (PD-L1) and tumor-associated macrophages are closely related to the development of lung cancer. However, whether tumor-derived exosomal PD-L1 could mediate the regulation of macrophage polarization in lung cancer remains unclear. For this research, the level of PD-L1 in normal tissues and lung cancer tissues was evaluated using RT-qPCR. Next, the apoptosis of lung cancer cells was evaluated using flow cytometry assay. Then, the structure and morphology of vesicles were observed using transmission electron microscopy and nanoparticle tracking analysis. Later on, the internalization of exosomes by macrophage was observed using fluorescence microscopy. Our results showed that the level of PD-L1 was upregulated in tumor tissues and lung cancer cells. Knockdown of PD-L1 notably inhibited the viability, migration and invasion of lung cancer cells. In addition, lung cancer cells-derived exosomal PD-L1 could be absorbed by macrophages. Meanwhile, exosomal PD-L1 was able to promote macrophages M2 polarization. Moreover, macrophages M2 polarization induced by exosomal PD-L1 further remarkably promoted the viability, migration, invasion, and epithelial-mesenchymal transition process of lung cancer cells. Collectively, knockdown of PD-L1 notably inhibited the viability, migration and invasion of lung cancer cells. Tumor cell-derived exosomal PD-L1 could promote the growth of lung cancer cells by mediating macrophages M2 polarization. Thus, inhibiting macrophages M2 polarization might be a promoting therapy for the treatment of lung cancer.

Primary pulmonary meningioma: A case report.

Meningioma is a common benign tumor originating in the brain, but primary pulmonary meningioma is very rare. It is usually benign, slow-growing, and responds well to treatment. The most common manifestation is an isolated pulmonary nodule that radiologically resembles a benign primary lung tumor. Herein, we report a case of a complete thoracoscopic resection of a primary pulmonary meningioma.

Spatial and temporal variations of net ecosystem productivity in Xinjiang Autonomous Region, China based on remote sensing.

Net ecosystem productivity (NEP), which plays a key role in the carbon cycle, is an important indicator of the ecosystem's carbon budget. In this paper, the spatial and temporal variations of NEP over Xinjiang Autonomous Region, China from 2001 to 2020 were studied based on remote sensing and climate re-analysis data. The modified Carnegie Ames Stanford Approach (CASA) model was employed to estimate net primary productivity (NPP), and the soil heterotrophic respiration model was used to calculate soil heterotrophic respiration. Then NEP was obtained by calculating the difference between NPP and heterotrophic respiration. The annual mean NEP of the study area was high in the east and low in the west, high in the north and low in the south. The 20-year mean vegetation NEP of the study area is 128.54 gC·m-2, indicating that the study area is a carbon sink on the whole. From 2001 to 2020, the annual mean vegetation NEP ranged between 93.12 and 158.05 gC·m-2, and exhibited an increasing trend in general. 71.46% of the vegetation area showed increasing trends of NEP. NEP exhibited a positive relationship with precipitation and a negative relationship with air temperature, and the correlation with air temperature was more significant. The work reveals the spatio-temporal dynamics of NEP in Xinjiang Autonomous Region and can provide a valuable reference for assessing regional carbon sequestration capacity.

The Electrical and Thermal Characteristics of Stacked GaN MISHEMT.

To study the working performance of 3D stacked chips, a double-layer stacked GaN MISHEMTs structure was designed to study the electro-thermal characteristics and heat transfer process of stacked chips. Firstly, the electrical characteristics of double-layer and single-layer GaN MISHEMTs are compared at room temperature. Under the same conditions, the output current of double-layer GaN MISHEMTs is twice that of single-layer GaN MISHEMTs, but its off-state current is much higher than that of a single-layer device. Meanwhile, there is no significant difference between the threshold voltages of the double-layer and single-layer GaN MISHEMTs. Then, the effect of temperature on the electrical characteristics of double-layer GaN MISHEMTs is also investigated. When the temperature increased from room temperature to 150 °C, the device's threshold voltage gradually shifted negatively, the output current of the device decreased, and the off-state current of the device increased. Furthermore, a thermal resistance network model has been established to analyze the thermal characteristics of the stacked GaN MISHEMTs. The relative error between the results calculated according to the model and the experimental results does not exceed 4.26%, which verified the correctness and accuracy of the presented model to predict the temperature distribution of the stacked GaN MISHEMTs.

Comparison Between PE-TLIF and MIS-TLIF in the Treatment of Middle-Aged and Elderly Patients with Single-Level Lumbar Disc Herniation.

Objective: To evaluate the early clinical effect of percutaneous endoscopic transforaminal lumbar interbody fusion (PE-TLIF) and minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) surgery in the treatment of middle-aged and elderly patients with single-level lumbar disc herniation accompanied by lumbar instability. Methods: From January 2019 to June 2020, a total of 82 consecutive patients were categorised into PE-TLIF group and MIS-TLIF group based on different surgical methods. The visual analog scale (VAS), Oswestry disability index (ODI), Japanese Orthopaedic Association (JOA) score, perioperative objective serological index, operation time, intraoperative blood loss, time to back to work or normal life, and Modified MacNab score were used as the evaluation indexes. The differences between the two groups were analyzed and the clinical effects were compared. Results: The VAS back pain of PE-TLIF group was decreased compared to that of MIS-TLIF group in the postoperative 1 week and 1 month. The operative time in PE-TLIF group was obviously longer than that in MIS-TLIF group. The hospital stay was significantly shorter in PE-TLIF group than that in MIS-TLIF group. More intraoperative blood loss and postoperative drainage were recorded in MIS-TLIF group. Compared with MIS-TLIF, PE-TLIF surgery was associated with a shorter time to ambulation after surgery and a shorter time to back to work or normal life. Significant statistical differences were observed in IL-6, CRP, and CK on postoperative 3 days between the two groups. Conclusion: For middle-aged and elderly patients, PE-TLIF and MIS-TLIF surgery both have obvious clinical efficacy and safety. However, with less intraoperative blood loss, shorter recovery time and less injury to the patients, people undergoing PE-TLIF surgery can return to work or normal life faster. It is speculated that PE-TLIF has a higher incidence of complications and recurrence rate than that MIS-TLIF. PE-TLIF may be a better choice for middle-aged and elderly patients with single-level lumbar disc herniation.

Down-regulation of CLEC3B facilitates epithelial-mesenchymal transition, migration and invasion of lung adenocarcinoma cells.

BACKGROUND: Lung adenocarcinoma (LUAD) belongs to non-small cell lung carcinoma, and the metastasis is the main cause of death in LUAD patients. It is generally accepted that cell adhesion is closely associated with tumor metastasis. Accordingly, the purpose of this study is to investigate the functions of CLEC3B to the invasion, adhesion, and migration of LUAD cells. METHODS: Through bioinformatic analysis, CLEC3B level was found markedly down-regulated in LUAD tissue. Parallel-plate flow chamber, wound-healing and Transwell assays were taken to detect the cell adhesion, invasion, and migration. qRT-PCR and western blot analyzed expression of CLEC3B, p53 and epithelial-mesenchymal transition (EMT) marker. RESULTS: When CLEC3B was lowly-expressed in LUAD cell lines, the cell adhesion capability was also lowered, with the EMT, migration and invasion of the cells progressed. Abnormal expression of CLEC3B was related to p53 signaling pathway. CONCLUSION: Above all, a further investigation of the function of CLEC3B in LUAD helps us further understand the molecular mechanism of the tumor metastasis.

Estimation of homocysteine concentration as an indicator of foetal death in pregnant Chinese women with preeclampsia: A case-control study.

INTRODUCTION: This study evaluated whether changes in homocysteine concentrations in pregnant women with preeclampsia (PE) might be useful for predicting foetal death. MATERIALS AND METHODS: This study evaluated 1,368 PE women at two Chinese centres. Medical records were reviewed to collect data regarding maternal age, homocysteine concentrations and other clinical parameters. RESULTS: Maternal serum homocysteine concentrations were significantly higher in the group with PE than control. Significant differences (p < 0.05) were also observed between the foetal death and survival groups in terms of body mass index, neonatal weight, previous deliveries, gestation length and adverse pregnancy history. Multivariate logistic regression analysis revealed that upper-quartile homocysteine concentration was a significant risk factor of foetal death in the group with PE, and overall survival rate of patients with high homocysteine concentrations during pregnancy was significantly lower than those with low level (p < 0.05). CONCLUSIONS: Our results indicate that foetal death was associated with upper-quartile homocysteine concentrations in the group with PE, it can be an indicator of foetal death throughout the pregnancy.

Genomic alteration profiles of lung cancer and their relationship to clinical features and prognosis value using individualized genetic testing.

BACKGROUND: This study aimed to use a panel targeting 197 genes and 38 fusions to observe the features of gene variations in lung cancer patients, as well as their prognostic values. METHODS: Patients admitted to our hospital between 2016 and 2017 were enrolled. All patients received OseqTM-Drug genetic testing using peripheral venous blood, followed by 1-2 years of observation. RESULTS: For all included patients, 32 genes were observed with mutations. EGFR exhibited the highest mutation rate (46.5%), followed by TP53. The majority of patients carried only one mutant gene. Interestingly, 18 (41.8%) patients showed no mutations, and some cases carried mutations in six genes simultaneously. There was no statistical relationship between mutations and demographic influence. Pathological subtypes were associated with mutations including FLI1, IGF1R, and NOTCH1. A significant correlation was observed between mutant genes and stage at diagnosis, however this requires further confirmation as there was only one case in these mutations: AKT2, AR, STK11, VEGFA, HDAC6, and ASPSCR. For the 33 patients with lymph node metastases at the time of diagnosis, no correlation with any gene mutant was found. Finally, no associations between the survival or prognosis indices (1-year survival, 1-year progression, progression free survival (PFS), and overall survival (OS)) were observed with gene mutations. CONCLUSIONS: Together, individualized genetic testing is a feasible and minimally invasive approach in cancer genetic analysis. However, gene mutation detection has a limited efficacy in the prediction of prognosis.

Multiscale Deficiency Integration by Na-Rich Engineering for High-Stability Li-Rich Layered Oxide Cathodes.

Lithium-rich manganese-based (LRM) layered oxides are considered as one of the most promising cathode materials for next-generation high-energy-density lithium-ion batteries (LIBs) because of their high specific capacity (>250 mAh g-1). However, they also go through severe capacity decay, serious voltage fading, and poor rate capability during cycling. Herein, a multiscale deficiency integration, including surface coating, subsurface defect construction, and bulk doping, is realized in a Li1.2Mn0.54Ni0.13Co0.13O2 cathode material by facile Na-rich engineering through a sol-gel method. This multiscale design can significantly improve the bulk and surface structural stability and diffusion rate of Li+ ions of electrode materials. Specifically, an outstanding specific capacity of 201 mAh g-1 is delivered at 1C of the designed cathode material after 400 cycles, relating to a large capacity retention of 89.0%. Meanwhile, the average voltage is retained up to 3.13 V with a large voltage retention of 89.6% and the energy density is maintained at 627.4 Wh kg-1. In situ X-ray diffraction (XRD), ex situ transmission electron microscopy (TEM) investigations, and density functional theory (DFT) calculations are conducted to explain the greatly enhanced electrochemical properties of a LRM cathode. We believe that this strategy would be a meaningful reference of LRM cathode materials for the research in the future.

Designing Uniformly Layered FeTiO3 Assemblies Consisting of Fine Nanoparticles Enabling High-Performance Quasi-Solid-State Sodium-Ion Capacitors.

Sodium-ion capacitors (NICs) that have integrated the dual advantages of the high output of supercapacitors and the high energy density of batteries have stimulated growing attention for the next generation of practical electrochemical energy storage devices. The last years have seen the unprecedentedly rapid emergence of ilmenite materials, which present great promise in the realm of energy storage. However, NICs based on ilmenite materials have been scarcely researched so far. Instead, most of the current devices explored applied flammable liquid electrolytes, leading to a concern about unexpected leakage and potential safety problems. Herein, a quasi-solid-state NIC is constructed by employing the prepared uniformly layered FeTiO3 assemblies consisting of fine nanoparticles as anode and sodium ion conducting gel polymer as electrolyte. The resulting device delivers a high-energy-high-power density (79.8 Wh kg-1, 6,750 W kg-1), putting it among the state-of-the-art NICs. Furthermore, the assembled quasi-solid-state device also manifests long-term cycling stability over 2,000 cycles with a capacity retention ~80%. The uniformly layered FeTiO3 has great potential in developing low-cost and high-performance electrodes for the next generation of sodium and other metal ions-based energy storage devices.

DSSR-enabled innovative schematics of 3D nucleic acid structures with PyMOL.

Sophisticated analysis and simplified visualization are crucial for understanding complicated structures of biomacromolecules. DSSR (Dissecting the Spatial Structure of RNA) is an integrated computational tool that has streamlined the analysis and annotation of 3D nucleic acid structures. The program creates schematic block representations in diverse styles that can be seamlessly integrated into PyMOL and complement its other popular visualization options. In addition to portraying individual base blocks, DSSR can draw Watson-Crick pairs as long blocks and highlight the minor-groove edges. Notably, DSSR can dramatically simplify the depiction of G-quadruplexes by automatically detecting G-tetrads and treating them as large square blocks. The DSSR-enabled innovative schematics with PyMOL are aesthetically pleasing and highly informative: the base identity, pairing geometry, stacking interactions, double-helical stems, and G-quadruplexes are immediately obvious. These features can be accessed via four interfaces: the command-line interface, the DSSR plugin for PyMOL, the web application, and the web application programming interface. The supplemental PDF serves as a practical guide, with complete and reproducible examples. Thus, even beginners or occasional users can get started quickly, especially via the web application at http://skmatic.x3dna.org.

Toward a Mechanistic Understanding of DNA Methylation Readout by Transcription Factors.

Epigenetic DNA modification impacts gene expression, but the underlying molecular mechanisms are only partly understood. Adding a methyl group to a cytosine base locally modifies the structural features of DNA in multiple ways, which may change the interaction with DNA-binding transcription factors (TFs) and trigger a cascade of downstream molecular events. Cells can be probed using various functional genomics assays, but it is difficult to disentangle the confounded effects of DNA modification on TF binding, chromatin accessibility, intranuclear variation in local TF concentration, and rate of transcription. Here we discuss how high-throughput in vitro profiling of protein-DNA interactions has enabled comprehensive characterization and quantification of the methylation sensitivity of TFs. Despite the limited structural data for DNA containing methylated cytosine, automated analysis of structural information in the Protein Data Bank (PDB) shows how 5-methylcytosine (5mC) can be recognized in various ways by amino acid side chains. We discuss how a context-dependent effect of methylation on DNA groove geometry can affect DNA binding by homeodomain proteins and how principled modeling of ChIP-seq data can overcome the confounding that makes the interpretation of in vivo data challenging. The emerging picture is that epigenetic modifications affect TF binding in a highly context-specific manner, with a direction and effect size that depend critically on their position within the TF binding site and the amino acid sequence of the TF. With this improved mechanistic knowledge, we have come closer to understanding how cells use DNA modification to acquire, retain, and change their identity.

Web 3DNA 2.0 for the analysis, visualization, and modeling of 3D nucleic acid structures.

Web 3DNA (w3DNA) 2.0 is a significantly enhanced version of the widely used w3DNA server for the analysis, visualization, and modeling of 3D nucleic-acid-containing structures. Since its initial release in 2009, the w3DNA server has continuously served the community by making commonly-used features of the 3DNA suite of command-line programs readily accessible. However, due to the lack of updates, w3DNA has clearly shown its age in terms of modern web technologies and it has long lagged behind further developments of 3DNA per se. The w3DNA 2.0 server presented here overcomes all known shortcomings of w3DNA while maintaining its battle-tested characteristics. Technically, w3DNA 2.0 implements a simple and intuitive interface (with sensible defaults) for increased usability, and it complies with HTML5 web standards for broad accessibility. Featurewise, w3DNA 2.0 employs the most recent version of 3DNA, enhanced with many new functionalities, including: the automatic handling of modified nucleotides; a set of 'simple' base-pair and step parameters for qualitative characterization of non-Watson-Crick double-helical structures; new structural parameters that integrate the rigid base plane and the backbone phosphate group, the two nucleic acid components most reliably determined with X-ray crystallography; in silico base mutations that preserve the backbone geometry; and a notably improved module for building models of single-stranded RNA, double-helical DNA, Pauling triplex, G-quadruplex, or DNA structures 'decorated' with proteins. The w3DNA 2.0 server is freely available, without registration, at http://web.x3dna.org.

DNA Conformational Changes Play a Force-Generating Role during Bacteriophage Genome Packaging.

Motors that move DNA, or that move along DNA, play essential roles in DNA replication, transcription, recombination, and chromosome segregation. The mechanisms by which these DNA translocases operate remain largely unknown. Some double-stranded DNA (dsDNA) viruses use an ATP-dependent motor to drive DNA into preformed capsids. These include several human pathogens as well as dsDNA bacteriophages-viruses that infect bacteria. We previously proposed that DNA is not a passive substrate of bacteriophage packaging motors but is instead an active component of the machinery. We carried out computational studies on dsDNA in the channels of viral portal proteins, and they reveal DNA conformational changes consistent with that hypothesis. dsDNA becomes longer ("stretched") in regions of high negative electrostatic potential and shorter ("scrunched") in regions of high positive potential. These results suggest a mechanism that electrostatically couples the energy released by ATP hydrolysis to DNA translocation: The chemical cycle of ATP binding, hydrolysis, and product release drives a cycle of protein conformational changes. This produces changes in the electrostatic potential in the channel through the portal, and these drive cyclic changes in the length of dsDNA as the phosphate groups respond to the protein's electrostatic potential. The DNA motions are captured by a coordinated protein-DNA grip-and-release cycle to produce DNA translocation. In short, the ATPase, portal, and dsDNA work synergistically to promote genome packaging.

Effects of Noncanonical Base Pairing on RNA Folding: Structural Context and Spatial Arrangements of G·A Pairs.

Noncanonical base pairs play important roles in assembling the three-dimensional structures critical to the diverse functions of RNA. These associations contribute to the looped segments that intersperse the canonical double-helical elements within folded, globular RNA molecules. They stitch together various structural elements, serve as recognition elements for other molecules, and act as sites of intrinsic stiffness or deformability. This work takes advantage of new software (DSSR) designed to streamline the analysis and annotation of RNA three-dimensional structures. The multiscale structural information gathered for individual molecules, combined with the growing number of unique, well-resolved RNA structures, makes it possible to examine the collective features deeply and to uncover previously unrecognized patterns of chain organization. Here we focus on a subset of noncanonical base pairs involving guanine and adenine and the links between their modes of association, secondary structural context, and contributions to tertiary folding. The rigorous descriptions of base-pair geometry that we employ facilitate characterization of recurrent geometric motifs and the structural settings in which these arrangements occur. Moreover, the numerical parameters hint at the natural motions of the interacting bases and the pathways likely to connect different spatial forms. We draw attention to higher-order multiplexes involving two or more G·A pairs and the roles these associations appear to play in bridging different secondary structural units. The collective data reveal pairing propensities in base organization, secondary structural context, and deformability and serve as a starting point for further multiscale investigations and/or simulations of RNA folding.

Mechanisms of silver nanoparticles-induced cytotoxicity and apoptosis in rat tracheal epithelial cells.

Silver nanoparticles (AgNPs) are increasingly utilized in a number of applications. This study was designed to investigate AgNPs induced cytotoxicity, oxidative stress and apoptosis in rat tracheal epithelial cells (RTE). The RTE cells were treated with 0, 100 µg/L and 10,000 µg/L of the AgNPs with diameters of 10 nm and 100 nm for 12 hr. The cell inhibition level, apoptosis ratio, reactive oxygen species (ROS), malondialdehyde (MDA) and metallothionein (MT) content were determined. The mRNA expression of cytoc, caspase 3, and caspase 9 was measured by quantitative real-time polymerase chain reaction (qRT-PCR). In addition, we also analyzed the cytoc, caspase 3, pro-caspase 3, caspase 9, and pro-caspase 9 protein expression by western blotting. Electric cell-substrate impedance sensing (ECIS) analysis showed that the growth and proliferation of RTE cells were significantly inhibited in a dose-dependent manner under AgNPs exposure. The cell dynamic changes induced by 10 nm AgNPs were more severe than that of the 100 nm AgNPs exposure group. The intracellular MT, ROS, and MDA content increased when the exposure concentration increased and size reduced, whereas Ca2+-ATPase activity and Na+/K+-ATPase activity changed inversely. The relative expression of protein of cytoc, caspase 3, and caspase 9 were upregulated significantly, which indicated that AgNPs induced apoptosis of RTE cells through the caspase-dependent mitochondrial pathway. Our results demonstrate that AgNPs caused obvious cytotoxicity, oxidative stress, and apoptosis in RTE cells, which promoted the releasing of cytochrome C and pro-apoptotic proteins into the cytoplasm to activate the caspase cascade and finally led to apoptosis.

Effects of Perfluorooctanoic Acid on the Associated Genes Expression of Autophagy Signaling Pathway of Carassius auratus Lymphocytes in vitro.

Perfluorooctanoic acid (PFOA) has been detected in various water bodies and caused harm to aquatic organisms. The aim of this study was to investigate the cytotoxicity and mechanism associated with autophagy and oxidative stress after exposure to PFOA (0, 1, 10, 100 µg/L) for 12 h on lymphocytes, which was isolated from the head kidney of Carassius auratus (C. auratus). Both of autophagy formation, cell activity, and intracellular reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) levels were measured. The relative expression of partial autophagy-related genes autophagy related 5 (Atg 5), autophagy related 7 (Atg 7), and Beclin 1 were also cloned and detected. Homologous relationships analysis showed high identities of genes in C. auratus and other fish by blast. C. auratus lymphocytes growth inhibition rates was increased induced by PFOA. Compared with the control group, the ROS generation and the MDA content were significantly increased in all of the PFOA-treated group. Besides, decreased SOD activity and decrease of GSH activity induced by PFOA further confirmed the occurrence of oxidative stress. The number of autophagosome formations was increased in a dose-dependent manner. Compared with the control group, Atg 7 and Beclin 1 mRNA expression was elevated significantly after PFOA exposed, showing a time-dependent manner, while mRNA expression of Atg 5 was increased remarkably in 100 µg/L PFOA-treated group. Our results indicated that PFOA caused oxidative damage to lymphocytes in C. auratus and caused various autophagy signaling pathway-associated genes imbalances in the lymphocytes. Autophagy signaling pathway-associated genes imbalance could weaken antioxidant capacity and involve in the mechanism of C. auratus lymphocytes oxidative injury caused by PFOA.

Role of the Nrf2-ARE pathway in perfluorooctanoic acid (PFOA)-induced hepatotoxicity in Rana nigromaculata.

Perfluorooctanoic acid (PFOA) is widely distributed in various environmental media and is toxic to organisms. This study demonstrated that PFOA induces hepatotoxicity in the frog and evaluated the role of CYP3A and the Nrf2-ARE signaling pathway in regulating responses to PFOA-induced hepatotoxicity. Rana nigromaculata were exposed to 0, 0.01, 0.1, 0.5, or 1 mg/L PFOA solutions in a static-renewal system for 14 days. Liver tissue samples were collected 24 h after the last treatment. Hepatic histology was observed by HE staining and transmission electron microscopy. The oxidative stress levels in the liver were measured. The expression levels of CYP3A, Nrf2, NQO1, and HO-1 mRNA were measured by quantitative reverse transcription-polymerase chain reaction. PFOA-treated frog liver tissue exhibited diffuse cell borders, cytoplasmic vacuolization, broken nuclei, nuclear chromatin margination, and swollen mitochondria. In addition, the livers of PFOA-treated frogs showed a significantly elevated content of reactive oxygen species, malondialdehyde, glutathione and glutathione S-transferase activity compared to the livers of control frogs. However, the glutathione peroxidase activities concomitantly decreased in PFOA-treated frogs compared to those in the control group. Furthermore, compared with control frogs, the expression levels of CYP3A, Nrf2, and NQO1 mRNA significantly increased in PFOA-treated frogs. HO-1 mRNA expression remarkably increased only in groups treated with 0.5 or 1 mg/L PFOA. Our results indicate that PFOA induces hepatotoxicity in a dose-dependent manner. Furthermore, the results of the comparison analysis between different gender groups illustrated that PFOA is more toxic to female frogs than male frogs. Our results demonstrated that PFOA causes liver damage and that CYP3A enhances PFOA-induced female frogs hepatotoxicity are more virulent than male through biotransformation, and the activation of the Nrf2-ARE pathway is induced to protect against hepatotoxicity in Rana nigromaculata, all of which provide the scientific basis for the protection of amphibians against environmental contaminants.