Grass carp (Ctenopharyngodon idella) deacetylase SIRT1 targets p53 to suppress apoptosis in a KAT8 dependent or independent manner.

Sirtuin1 (SIRT1) is known as a deacetylase to control various physiological processes. In mammals, SIRT1 inhibits apoptotic process, but the detailed mechanism is not very clear. Here, our study revealed that grass carp (Ctenopharyngodon idella) SIRT1 (CiSIRT1, MN125614.1) inhibits apoptosis through targeting p53 in a KAT8-dependent or a KAT8-independent manner. In CIK cells, CiSIRT1 over-expression results in significant decrease of some apoptotic gene expressions, including Bax/Bcl2, caspase3 and caspase9, whereas CiKAT8 or Cip53 facilitates the induction of apoptosis. Because CiSIRT1 separately interacted with CiKAT8 and Cip53, we speculated that CiSIRT1 blocked apoptosis may be by virtue of KAT8-p53 axis or directly by p53. In a KAT8-dependent manner, CiSIRT1 interacted with CiKAT8, then reduced the acetylation of CiKAT8 and subsequently promoted its degradation. Then, CiKAT8 acetylated p53 and induced p53-mediated apoptosis. MYST domain of CiKAT8 was critical in this pathway. In a KAT8-independent manner, CiSIRT1 also inhibited p53-induced apoptosis by directly deacetylating p53 and promoting the degradation of p53. Generally, these findings uncovered two pathways in which CiSIRT1 decreases the acetylation of p53 via a KAT8-dependent or a KAT8-independent manner.

Grass carp (Ctenopharyngodon idella) NIK up-regulates the expression of IL-8 by activating the NF-κB canonical pathway.

NIK (NF-κB inducing kinase) belongs to the mitogen-activated protein kinase family, which activates NF-κB and plays a vital role in immunology, inflammation, apoptosis, and a series of pathological responses. In NF-κB noncanonical pathway, NIK and IKKα have been often studied in mammals and zebrafish. However, few have explored the relationship between NIK and other subunits of the IKK complex. As a classic kinase in the NF-κB canonical pathway, IKKß has never been researched with NIK in fish. In this paper, the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) NIK (CiNIK) was first cloned and identified. The expression level of CiNIK in grass carp cells was increased under GCRV stimuli. Under the stimulation of GCRV, poly (I:C), and LPS, the expression of NIK in various tissues of grass carp was also increased. This suggests that CiNIK responds to viral stimuli. To study the relationship between CiNIK and CiIKKß, we co-transfected CiNIK-FLAG and CiIKKB-GFP into grass carp cells in coimmunoprecipitation and immunofluorescence experiments. The results revealed that CiNIK interacts with CiIKKß. Besides, the degree of autophosphorylation of CiNIK was enhanced under poly (I:C) stimulation. CiIKKß was phosphorylated by CiNIK and then activated the activity of p65. The activity change of p65 indicates that NF-κB downstream inflammatory genes will be functioning. CiNIK or CiIKKß up-regulated the expression of IL-8. It got higher when CiNIK and CiIKKß coexisted. This paper revealed that NF-κB canonical pathway and noncanonical pathway are not completely separated in generating benefits.

A molecular mechanism underlies grass carp (Ctenopharyngodon idella) TARBP2 regulating PKR-mediated cell apoptosis.

Interferon-inducible double-stranded RNA-dependent protein kinase (PKR) is one of the key antiviral arms in the innate immune system. The activated PKR performs its antiviral function by inhibiting protein translation and inducing apoptosis. In our previous study, we identified grass carp TARBP2 as an inhibitor of PKR activity, thereby suppressing cell apoptosis. This study aimed to explore the effects of grass carp TARBP2 on PKR activity and cell apoptosis. Grass carp TARBP2 comprises two N-terminal dsRBDs and a C-terminal C4 domain. Subcellular localization analysis conducted in CIK cells revealed that TARBP2-FL (full-length TARBP2), TARBP2-Δ1 (lack of the first dsRBD), and TARBP2-Δ2 (lack of the second dsRBD) are predominantly located in the cytoplasm, while TARBP2-Δ3 (lack of the two dsRBDs) is distributed both in the nucleus and cytoplasm. Colocalization and immunoprecipitation assays confirmed the interaction of TARBP2-FL, TARBP2-Δ1, and TARBP2-Δ2 with PKR, while TARBP2-Δ3 showed no binding. Furthermore, our findings suggested that the inhibitory effect of TARBP2-Δ1 or TARBP2-Δ2 on the PKR-eIF2α pathway is depressed compared to TARBP2-FL. In cell apoptosis assays, it was observed that TARBP2-FL inhibits PKR-mediated cell apoptosis. TARBP2-Δ1 or TARBP2-Δ2 exhibits decreased inhibition to PKR-mediated cell apoptosis, whereas TARBP2-Δ3 nearly completely loses this inhibitory effect. These findings highlight the critical importance of two dsRBDs of TARBP2 in interaction with PKR, as well as in the inhibition of PKR activity, resulting in the suppression of cell apoptosis triggered by prolonged PKR activation.

Succinate-induced macrophage polarization and RBP4 secretion promote vascular sprouting in ocular neovascularization.

Pathological neovascularization is a pivotal biological process in wet age-related macular degeneration (AMD), retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR), in which macrophages (Mφs) play a key role. Tip cell specialization is critical in angiogenesis; however, its interconnection with the surrounding immune environment remains unclear. Succinate is an intermediate in the tricarboxylic acid (TCA) cycle and was significantly elevated in patients with wet AMD by metabolomics. Advanced experiments revealed that SUCNR1 expression in Mφ and M2 polarization was detected in abnormal vessels of choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR) models. Succinate-induced M2 polarization via SUCNR1, which facilitated vascular endothelial cell (EC) migration, invasion, and tubulation, thus promoting angiogenesis in pathological neovascularization. Furthermore, evidence indicated that succinate triggered the release of RBP4 from Mφs into the surroundings to regulate endothelial sprouting and pathological angiogenesis via VEGFR2, a marker of tip cell formation. In conclusion, our results suggest that succinate represents a novel class of vasculature-inducing factors that modulate Mφ polarization and the RBP4/VEGFR2 pathway to induce pathological angiogenic signaling through tip cell specialization.

Grass carp (Ctenopharyngodon idella) Mex3B positively regulates innate immunity by promoting the K63-linked ubiquitination of TLR3.

As a member of Mex3 (muscle excess protein-3) family, Mex3B (Mex-3 RNA binding family member B) is crucial in cell proliferation and migration in mammals. In this study, an ortholog of mammalian Mex3B (denominated CiMex3B, MT276802.1) was cloned and identified in grass carp (Ctenopharyngodon idella). CiMex3B is 1578 bp in length and encodes a polypeptide of 525 amino acids. Consistent with its mammalian counterpart, CiMex3B also contains one C-terminal RING domain and two N-terminal conserved tandem KH domains. CiMex3B up-regulates the expressions of IFN1, ISG15, MX2, as well as the expressions of inflammatory cytokines such as IL6, IL8 and TNFα in response to poly(I:C). A screening test for identifying potential targets indicated that CiMex3B is associated with TLR3 and TRIF. CiMex3B co-localizes with TLR3 in the late endosome, mitochondria and endoplasmic reticulum after poly(I:C) stimulation, whereas they are rarely discovered in the lysosomes. CiMex3B serves as a positive regulator in the phosphorylation of IRF3 and induces IFN1 expression. In addition, two truncation mutants of CiMex3B (1-220 and 221-525) were constructed to better understand the molecular mechanism of CiMex3B-mediated ubiquitination of TLR3. In line with wild-type protein, CiMex3B mutant (1-220) was found mainly in the cytoplasm; however, CiMex3B mutant (221-525) resided in the cytoplasm and the nucleus as well, and it was further confirmed that CiMex3B mutant (221-525) still interacts with TLR3. We also observed that CiMex3B promotes the K63-linked ubiquitination of TLR3, while neither of the truncation mutants (1-220 or 221-525) retains this activity. To sum up, this study revealed that CiMex3B potentiates the K63-linked ubiquitination of TLR3, and then elicits the IRF3-mediated antiviral innate immune responses.

PM2.5 exposure increases dry eye disease risks through corneal epithelial inflammation and mitochondrial dysfunctions.

Dry eye disease (DED) is the most common disease affecting vision and quality of life. PM2.5 was a potential risk of DED. Herein, we conducted animal exposure and cell-based studies to evaluate the pathogenic effect of PM2.5 exposure on the ocular surface and DED etiological mechanisms. C57 mice were exposed to filtered air and PM2.5 aerosol. We assessed health conditions and inflammation of the ocular surface by corneal fluorescein staining and immunohistochemistry. In parallel, cultured human corneal epithelial cells (HCETs) were treated with PM2.5, followed by characterization of cell viability, intracellular ATP level, mitochondrial activities, and expression level of DED relevant mRNA and proteins. In mice, PM2.5 exposure induced severe superficial punctate keratopathy and inflammation in their cornea. In HCETs, cell proliferation and ROS generation followed dose-response and time-dependent manner; meanwhile, mitochondrial ROS (mtROS) level increased and mitochondrial membrane potential (MMP) level decreased. Inflammation cascade was triggered even after short-term exposure. The reduction of ATP production was alleviated with Nrf2 overexpression, NF-κB P65 knockdown, or ROS clearance. Nrf2 overexpression and P65 knockdown reduced inflammatory reaction through decreasing expression of P65 and increasing of Nrf2, respectively. They partly alleviated changes of ROS/mtROS/MMP. This research proved that PM2.5 would cause DED-related inflammation reaction on corneal epithelial cells and further explored its mechanism: ROS from mitochondrial dysfunctions of corneal epithelial cells after PM2.5 exposure partly inhibited the expression of anti-inflammatory protein Nrf2 led the activation of inflammatory protein P65 and its downstream molecules, which finally caused inflammation reaction.

Multi-dimensional evaluation of cardiotoxicity in mice following respiratory exposure to polystyrene nanoplastics.

BACKGROUND: Nanoplastics (NPs) could be released into environment through the degradation of plastic products, and their content in the air cannot be ignored. To date, no studies have focused on the cardiac injury effects and underlying mechanisms induced by respiratory exposure to NPs. RESULTS: Here, we systematically investigated the cardiotoxicity of 40 nm polystyrene nanoplastics (PS-NPs) in mice exposed via inhalation. Four exposure concentrations (0 µg/day, 16 µg/day, 40 µg/day and 100 µg/day) and three exposure durations (1 week, 4 weeks, 12 weeks) were set for more comprehensive information and RNA-seq was performed to reveal the potential mechanisms of cardiotoxicity after acute, subacute and subchronic exposure. PS-NPs induced cardiac injury in a dose-dependent and time-dependent manner. Acute, subacute and subchronic exposure increased the levels of injury biomarkers and inflammation and disturbed the equilibrium between oxidase and antioxidase activity. Subacute and subchronic exposure dampened the cardiac systolic function and contributed to structural and ultrastructural damage in heart. Mechanistically, violent inflammatory and immune responses were evoked after acute exposure. Moreover, disturbed energy metabolism, especially the TCA cycle, in the myocardium caused by mitochondria damage may be the latent mechanism of PS-NPs-induced cardiac injury after subacute and subchronic exposure. CONCLUSION: The present study evaluated the cardiotoxicity induced by respiratory exposure to PS-NPs from multiple dimensions, including the accumulation of PS-NPs, cardiac functional assessment, histology observation, biomarkers detection and transcriptomic study. PS-NPs resulted in cardiac injury structurally and functionally in a dose-dependent and time-dependent manner, and mitochondria damage of myocardium induced by PS-NPs may be the potential mechanism for its cardiotoxicity.

Chlorphoxim induces neurotoxicity in zebrafish embryo through activation of oxidative stress.

It is known that chlorphoxim is a broad-spectrum and high-effective pesticide. With the wide use in agricultural practice, chlorphoxim residue is also frequently detected in water, but its potential toxicity to aquatic life is still unclear. In this study, zebrafish is used as a model to detect the toxicity of chlorphoxim. Our results showed that exposure of high concentration of chlorphoxim at 96 h post-fertilization (hpf) resulted in a high mortality and pericardium edema rate, a low hatchability rate and heart rate. The nervous system damage, swimming behavior alteration and acetylcholinesterase (AChE) inhibition were measured in zebrafish embryos after a 6 days post-fertilization (dpf) of chlorphoxim exposure. The expression of neural-related genes is abnormal in zebrafish embryos. Chlorphoxim exposure significantly increases oxidative stress in zebrafish embryos by inhibiting antioxidant enzyme (SOD and CAT) and activating reactive oxygen species (ROS). As expected, chlorphoxim exposure induces apoptosis by enhancing the expression of apoptotic genes (Bax, Bcl2, and p53). Astaxanthin (ATX), an effective antioxidant, was found to be able to rescue the neurotoxicity of chlorphoxim through relieving oxidative stress and apoptosis. Altogether, the results showed that chlorphoxim exposure led to severe neurotoxicity to zebrafish embryos, which was contributed to a more comprehensive understanding of the safety use of the organophosphorus pesticide.

Grass carp (Ctenopharyngodon idella) NLK2 inhibits IFN I response through blocking MAVS-IRF3 axis.

In mammals, nemo-like kinase 2 (NLK2) is a conservative protein kinase involved in Wnt/ß-catenin signaling pathway and immune response. However, the role of NLK2 in immune response in teleost remain unclear. In this study, we identified an ortholog of mammalian NLK from grass carp (Ctenopharyngodon idellus) named CiNLK2. CiNLK2 shares a high level of homology with the counterparts, especially with that of Cyprinus carpio. CiNLK2 was ubiquitously expressed in all tested tissues (liver, brain, spleen, gill, kidney and eye) and its expression was up-regulated under the treatment with poly I:C or GCRV. Overexpression of CiNLK2 suppressed the production of IFN I in CIK cells whether or not treated with poly I:C. However, knockdown of CiNLK2 increased the expression level of IFN I. The analysis of subcellular localization showed that CiNLK2 protein was scattered throughout the cytoplasm and nucleus. In terms of mechanism, CiNLK2 can directly interact with MAVS and inhibit MAVS-induced IFN I response. Moreover, CiNLK2 increased the phosphorylation level of MAVS, which led to the degradation of MAVS protein. On the other hand, CiNLK2 suppressed the phosphorylation and nuclear translocation of IRF3. In general, CiNLK2 served as an inhibitor for IFN I response by targeting MAVS-IRF3 signal axis.

Grass carp (Ctenopharyngodon idella) DYRK2 modulates cell apoptosis through phosphorylating p53.

In mammals, DYRK2 increases p53 phosphorylation level by interacting with it and then promotes cell apoptosis. However, the function of fish DYRK2 has not yet been elucidated. In this paper, we cloned and identified the coding sequence (CDS) of a grass carp DYRK2 (CiDYRK2) which is 1773 bp in length and encodes 590 amino acids. SMART predictive analysis showed that CiDYRK2 possesses a serine/threonine kinase domain. Subsequently, we used the dsRNA analog polyinosinic-polycytidylic acid (poly (I:C) and Grass carp reovirus (GCRV) to stimulate grass carp and CIK cells for different times and found that CiDYRK2 mRNA was significantly up-regulated both in fish tissues and cells. To explore the function of CiDYRK2, we carried out overexpression and knockdown experiments of CiDYRK2 in CIK cells. Real-time quantitative PCR (Q-PCR), TdT-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry were used to detect the ratio of BAX/BCL-2 mRNA, the number of TUNEL positive cells, the proportion of Annexin V-positive cells respectively. The results showed that CiDYRK2 significantly up-regulated BAX/Bcl-2 mRNA ratio and increased the number of TUNEL-positive cells, as well as the proportion of Annexin V-positive cells. On the contrary, knock-down of CiDYRK2 significantly down-regulated BAX/Bcl-2 mRNA ratio in the cells. Therefore, CiDYRK2 promoted cell apoptosis. To study the molecular mechanism by which CiDYRK2 promoting cell apoptosis, subcellular localization and immunoprecipitation experiments were used to study the relationship between grass carp DYRK2 and the pro-apoptotic protein p53. The results showed that CiDYRK2 and Cip53 were located and co-localized in the nucleus. Co-immunoprecipitation experiment also showed that CiDYRK2 and Cip53 can bind with each other. We further found that DYRK2 can increase the phosphorylation level of p53. In a word, our results showed that grass carp DYRK2 induces cell apoptosis by increasing the phosphorylation level of p53.

Isoprocarb causes neurotoxicity of zebrafish embryos through oxidative stress-induced apoptosis.

Isoprocarb is a widely used carbamate insecticide in agriculture and aquaculture. Overuse of isoprocarb always leaves toxic residues in soil and water, however, the potential ecotoxicity of isoprocarb to organisms is still confusing. In this study, zebrafish embryo was used as a model to evaluate the toxicity of isoprocarb. Zebrafish embryos (96 hpf) were separately exposed at different concentrations of isoprocarb. The mortality rate, hatchability rate, average heart beat of the zebrafish embryo were separately calculated. Our results suggested that exposure to isoprocarb induced developmental toxicity in zebrafish embryos. HE staining showed that exposure to isoprocarb caused developmental defect in the hindbrain of zebrafish embryos. As expected, the behavioral analysis also showed that the motor ability of zebrafish embryos were significantly inhibited following exposure to isoprocarb. In terms of mechanism, The expressions of genes involved in neurodevelopment signaling pathways, such as foxo3a, gfap, syn2a, elavl3 and sox19b, were inhibited in zebrafish embryos after exposure to isoprocarb. The acetylcholinesterase (AChE) activity was also reduced in isoprocarb-treated zebrafish embryos. Moreover, oxidative stress was induced by increasing the reactive oxygen species (ROS) level and decreasing the activity of antioxidant enzyme (SOD) after exposure to isoprocarb. Expectedly, acridine orange (AO) staining and the detection of some apoptosis-related genes revealed that oxidative stress resulted in apoptosis. In short, the expressions of genes associated with the neurodevelopmental signaling pathway are inhibited, and oxidative stress is also induced in zebrafish embryos after exposure to isoprocarb, which may be the molecular basics of isoprocarb-induced neurotoxicity in zebrafish embryos.

Domain Knowledge-Based Evolutionary Reinforcement Learning for Sensor Placement.

Reducing pollutant detection time based on a reasonable sensor combination is desirable. Clean drinking water is essential to life. However, the water supply network (WSN) is a vulnerable target for accidental or intentional contamination due to its extensive geographic coverage, multiple points of access, backflow, infrastructure aging, and designed sabotage. Contaminants entering WSN are one of the most dangerous events that may cause sickness or even death among people. Using sensors to monitor the water quality in real time is one of the most effective ways to minimize negative consequences on public health. However, it is a challenge to deploy a limited number of sensors in a large-scale WSN. In this study, the sensor placement problem (SPP) is modeled as a sequential decision optimization problem, then an evolutionary reinforcement learning (ERL) algorithm based on domain knowledge is proposed to solve SPP. Extensive experiments have been conducted and the results show that our proposed algorithm outperforms meta-heuristic algorithms and deep reinforcement learning (DRL).

Diflubenzuron Induces Cardiotoxicity in Zebrafish Embryos.

Diflubenzuron is an insecticide that serves as a chitin inhibitor to restrict the growth of many harmful larvae, including mosquito larvae, cotton bollworm and flies. The residue of diflubenzuron is often detected in aquaculture, but its potential toxicity to aquatic organisms is still obscure. In this study, zebrafish embryos (from 6 h to 96 h post-fertilization, hpf) were exposed to different concentrations of diflubenzuron (0, 0.5, 1.5, 2.5, 3.5 and 4.5 mg/L), and the morphologic changes, mortality rate, hatchability rate and average heart rate were calculated. Diflubenzuron exposure increased the distance between the venous sinus and bulbar artery (SV-BA), inhibited proliferation of myocardial cells and damaged vascular development. In addition, diflubenzuron exposure also induced contents of reactive oxygen species (ROS) and malondialdehyde (MDA) and inhibited the activity of antioxidants, including SOD (superoxide dismutase) and CAT (catalase). Moreover, acridine orange (AO) staining showed that diflubenzuron exposure increased the apoptotic cells in the heart. Q-PCR also indicated that diflubenzuron exposure promoted the expression of apoptosis-related genes (bax, bcl2, p53, caspase3 and caspase9). However, the expression of some heart-related genes were inhibited. The oxidative stress-induced apoptosis damaged the cardiac development of zebrafish embryos. Therefore, diflubenzuron exposure induced severe cardiotoxicity in zebrafish embryos. The results contribute to a more comprehensive understanding of the safety use of diflubenzuron.

Fenpropathrin exposure induces neurotoxicity in zebrafish embryos.

Fenpropathrin has been a commonly used insecticide to control agricultural and household insects over a few decades. Up to now, fenpropathrin residue in soil and water has been often determined due to its widespread use, which poses serious threat to environment and aquatic organisms. The potential of fenpropathrin to affect aquatic lives is still poorly understood. In this study, we used zebrafish (Danio rerio) embryo as an experimental model system to evaluate the toxicity of fenpropathrin to the development of zebrafish nervous system. Zebrafish embryos were separately exposed to fenpropathrin at the dose of 0.016 mg/L, 0.032 mg/L, 0.064 mg/L, starting at 6 h post-fertilizationhpf (hpf) up to 96 hpf. The results showed that fenpropathrin exposure gives rise to physiological, behavioral, and neurodevelopmental impairments in zebrafish embryos, including enhanced acetylcholinesterase (AChE) activity, abnormal swimming behavior, karyopyknosis in brain cells, increased intercellular space, and uneven migration of neuron in brain area. In addition, the expressions of genes concerning neurodevelopment and neurotransmitter system were inhibited following fenpropathrin exposure. We also found that fenpropathrin exposure distinctly induced oxidative stress by increasing reactive oxygen species (ROS) generation and inhibiting the production of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). Expectedly, some apoptosis-associated genes were induced and the apoptosis appeared in the brain and heart cells of zebrafish embryos. Moreover, fenpropathrin exposure also inhibited the expressions of genes in Nrf2 signaling pathway, such as heme oxygenase-1 (HO-1) and SOD. In summary, the results of this study indicate that oxidative stress-triggered apoptosis may be an underlying fundamental of fenpropathrin-induced neurotoxicity in zebrafish embryos.

Grass carp (Ctenopharyngodon idellus) SHP2 suppresses IFN I expression via decreasing the phosphorylation of GSK3ß in a non-contact manner.

As a tyrosine phosphatase, Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) serves as an inhibitor in PI3K-Akt pathway. In mammals, SHP2 can phosphorylate GSK3ß at Y216 site to control the expression of IFN. So far, the multiple functions of SHP2 have been reported in mammals. However, little is known about fish SHP2. In this study, we cloned and identified a grass carp (Ctenopharyngodon idellus) SHP2 gene (CiSHP2, MT373151). SHP2 is conserved among different vertebrates by amino acid sequences alignment and the phylogenetic tree analysis. CiSHP2 shared the closest homology with Danio rerio SHP2. Simultaneously, SHP2 was also tested in grass carp tissues and CIK (C. idellus kidney) cells. We found that it responded to poly I:C stimulation. CiSHP2 was located in the cytoplasm just as the same as those of mammals. Interestingly, it inhibited the phosphorylation level of GSK3ß in a non-contact manner. Meanwhile CiGSK3ß interacted with and directly phosphorylated CiTBK1. In addition, we found that CiSHP2 also reduced the phosphorylation level of CiTBK1 by CiGSK3ß, and then it depressed the expression of IFN I via GSK3ß-TBK1 axis. These results suggested that CiSHP2 was involved in CiGSK3ß and CiTBK1 activity but not regulated their transcriptional level. At the same time, we also found that CiSHP2 also influenced the activity of CiIRF3. Therefore, fish SHP2 inhibited IFN I expression through blocking GSK3ß-TBK1 signal axis.

Ethoprophos induces cardiac toxicity in zebrafish embryos.

Ethoprophos is an effective and widely pesticide that used in controlling nemathelminth and soil insect. However, ethoprophos has been frequently detected in environment and freshwater. The potential toxicity to aquatic organisms is still not be explored. In this study, zebrafish embryo model was used to evaluated the toxicity of ethoprophos during cardiovascular developmental process of zebrafish. Zebrafish embryos were separately exposed to 10 mg/L, 20 mg/L, 30 mg/L, 40 mg/L and 50 mg/L of ethoprophos exposure at 96 h post-fertilization (hpf), which induced cardiac defects, such as low heart rate, pericardium edema and long SV-BA distance, but had no influence to vascular development. Mechanistically, the expression of cardiac-related genes were abnormal. Moreover, ethoprophos exposure significantly increased oxidative stress in zebrafish embryos by inhibiting the production of antioxidant enzyme (SOD) and activating reactive oxygen species. Expectedly, some apoptosis genes were induced and the apoptotic cardiomyocytes were detected by acridine orange staining. In addition, ethoprophos exposure also inhibited the expression of genes in wnt signaling pathway, such as ß-catenin, Axin2, GSK3ß and Sox9b. BML284, an activator of wnt signaling pathway, can rescue the cardiotoxic effect of embryos. These results indicated that oxidative stress and blocking wnt signaling pathway were molecular basis of ethoprophos-induced injure in zebrafish. Generally, our study showed that ethoprophos exposure led to severe cardiotoxicity to zebrafish embryo.

Grass carp (Ctenopharyngodon idella) IRAK1 and STAT3 up-regulate synergistically the transcription of IL-10.

In vertebrates, IL-10 is an anti-inflammatory factor that serves as a key inhibitory role in a wide range of immune responses. IRAK1 (IL-1 receptor-associated kinase 1), a key molecule in the inflammatory signal of IL-1R/TLR, plays an important pivotal role in regulating the autoimmunity of body. STAT3 (Signal transducer and activator of transcription 3) activated by IRAK1 participates in inflammation, tumorigenesis, metabolic disorders and immune response. Under the stimulation of LPS, IRAK1 enters the nucleus to form a dimer with STAT3 and regulates the expression of IL-10. However, the relationship between fish IRAK1 and STAT3 has not been reported. To explain the anti-inflammation in fish, we amplified and identified the complete open reading frame of grass carp IRAK1 (CiIRAK1) and STAT3 (CiSTAT3) based on the existing sequences. The expression of CiIRAK1 and CiSTAT3 were up-regulated significantly under the stimulation of LPS. This result suggests that both CiIRAK1 and CiSTAT3 may be involved in LPS-induced TLR4 pathway. The subcellular localization experiment revealed that CiIRAK1 is distributed in cytoplasm and enters nucleus after LPS stimulation. CiSTAT3 is distributed in both cytoplasm and nucleus with or without LPS stimulation. Immunoprecipitation assay revealed that CiIRAK1 interacted with CiSTAT3 under LPS stimulation. However in absence of LPS stimulation, CiIRAK1 and CiSTAT3 cannot interact with each other. Subsequently, immunofluorescence colocalization experiment further proved the interaction of CiIRAK1 and CiSTAT3 in nucleus under LPS stimulation. The dual luciferase reporter assays indicated that the binding of CiIRAK1 and CiSTAT3 synergistically enhanced the activity of CiIL-10 promoter.

Grass carp (Ctenopharyngodon idella) PACT induces cell apoptosis and activates NF-кB via PKR.

As a dsRNA-dependent and interferon-induced protein kinase, PKR is involved in antiviral immune response and apoptosis mediated by various cytokines. In mammalian cells, PKR can also be activated in the absence of dsRNA. A PKR activator, PACT (PKR activating protein), also referred to as RAX (PKR-associated protein X) plays an important role. In recent years, with the increasing recognition of fish interferon system, PKR and PACT have been gradually revealed in fish. However, the function of fish PACT is unclear. In our previous work, we suggested that grass carp (Ctenopharyngodon idella) PACT must be involved in IRF2 and ATF4-mediated stress response pathways. In the present study, we found that the expression of C. idella PACT (CiPACT) and CiPKR were significantly up-regulated under the stimulation of LPS. It indicated that CiPACT and CiPKR may play an important role in response to LPS stimulation. In addition, the response time of CiPACT to LPS is earlier than that of CiPKR. It has also shown that overexpression of CiPACT in CIK cells can significantly enhance the level of p-eIF2α, induces apoptosis and translocation of Cip65 to nucleus from cytoplasm. To further understand the mechanism, we carried out the co-immunoprecipitation assay. It proved that the interaction of CiPACT and CiPKR made the phosphorylation of CiPKR. Overexpression of CiPACT induced the down-regulation of intracellular expression of bcl-2 and up-regulation of bax. However, in CiPKR knocked-down cells the expression of bcl-2 and bax were just the opposite. Therefore, the mechanism of fish PACT induces apoptosis and activates NF-кB is dependent on PKR.

Effects of spinetoram on the developmental toxicity and immunotoxicity of zebrafish.

Our study investigated the effects of spinetoram on the developmental toxicity and immunotoxicity of zebrafish. 10 h post-fertilization (hpf) zebrafish embryos were exposed to several concentrations of spinetoram (0, 5.0 mg/L, 7.5 mg/L, 10 mg/L) for up to 96 hpf, and their mortality, heart rate, number of innate and adaptive immune cells, oxidative stress, apoptosis and gene expression were detected. Studies indicated that the spinetoram exposed zebrafish embryos showed yolk sac edema, slow growth, decreased heart rate, decreased number of immune cells, delayed thymic development and cell apoptosis. In addition, there were also significant changes in oxidative stress related indicators in zebrafish, the content of ROS and MDA and the activity of CAT and SOD increased with the increase of spinetoram concentration. Moreover, we detected the expression of TLR4 related genes including TLR4, MYD88 and NF-κB p65 which were significantly up-regulated in the treated groups. Meanwhile, we also found that pro-inflammatory factors IL-6, IL-8, IFN-γ and CXCL-c1c were up-regulated, but anti-inflammatory factor IL-10 was down-regulated in the treated groups. Briefly, our results show that spinetoram induces the developmental toxicity and immunotoxicity of zebrafish to a certain extent, providing basis for the further research on the molecular mechanism of spinetoram exposure to aquatic ecosystems.

IRF9 promotes apoptosis and innate immunity by inhibiting SIRT1-p53 axis in fish.

As a NAD+-dependent deacetylase, SIRT1 is widely involved in apoptosis and cellular inflammation via multiple pathways such as p53, NF-кB and STAT. More and more studies have shown that p53 is the first non-histone deacetylation target of SIRT1. SIRT1-p53 axis thus plays an important role in mammalian cells. IRF9 is an important member of interferon regulator factor family and performs an important role in innate immunity against foreign virus invasion. More importantly, human IRF9 can suppress the SIRT1-p53 axis. However, the functions and relationship between IRF9 and SIRT1-p53 axis are rarely studied in fish. To this end, we made a preliminary research on the functions of grass carp (Ctenopharyngodon idella) IRF9, SIRT1 and p53 in apoptosis and innate immunity. Firstly, we cloned and identified the ORF of SIRT1 (named CiSIRT1, MN125614) from C. idella and demonstrated that CiIRF9 promoted apoptosis, while CiSIRT1 inhibited apoptosis by flow cytometry and TUNEL experiments. Next, we found the interaction between CiSIRT1 and Cip53 in vivo by co-immunoprecipitation experiments. Moreover, the colocalization analysis also showed CiSIRT1 and Cip53 were mainly distributed in nucleus. Thirdly, we got a conclusion that CiIRF9 can repress the expression of CiSIRT1, implying that CiIRF9 regulates CiSIRT1-p53 axis. Finally, CiSIRT1 mRNA level was significantly up-regulated and the expression reached the highest level at 24 h post poly (I:C) stimulation in CIK cells. So, CiSIRT1 may exert an important function in innate immunity. Furthermore, we found CiSIRT1 down-regulated the expression of CiIFN1. In summary, CiIRF9 promotes apoptosis and innate immunity by inhibiting SIRT1-p53 axis. These findings will provide a new theoretical basis for the research on teleost innate immunity.