Comparative proteomic analysis of wild-type and a SlETR-3 (Nr) mutant reveals an ethylene-induced physiological regulatory network in fresh-cut tomatoes.

Ethylene plays a crucial role in regulating fruit ripening, quality, and defense response. However, the mechanism(s) responsible for wound-induced ethylene regulation of fruit physiology at a network level is unclear. We used mass spectrometry (MS) to identify differences in the physiological response between fresh-cut fruits of wild-type (WT) tomato and an ethylene receptor mutant (SlETR-3) (also referred to as Nr) during storage. We found that Nr mutants exhibited better appearance and quality, as well as higher ethylene levels during the first 3 d of storage at 4 °C. Thirty-seven (0 h), eighty-two (12 h) and twelve (24 h) differentially abundant proteins were identified between the fresh-cut slices of the two genotypes during storage at the designated timepoints. In particular, antioxidant enzymes, such as ascorbate peroxidase, glutathione S-transferase, and peroxiredoxin were highly expressed in WT fruit, which was associated with higher H2O2 production, and high levels of transcription of cell-wall degrading enzymes. Leucine aminopeptidase, a marker enzyme for response to wounding exhibited higher levels in the Nr mutant, which is consistent with its higher production of ethylene. Collectively, our results provide a deeper insight into the ethylene-induced physiological regulatory network that is activated in fresh-cut tomatoes.

Differentiation therapy: Unlocking phenotypic plasticity of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. The current treatment of HCC mainly includes surgery, chemotherapy, and liver transplantation. HCC differentiation therapy aims to restore tumor cells' normal liver characteristics and unlock their phenotypic plasticity. Understanding the molecular and signaling pathways that control HCC differentiation can help identify new targets for inducing differentiation and provide ideas for drug design. Downregulation of liver enriched transcription factors, imbalanced signal pathway, and dysregulated microRNA play essential roles in regulating the differentiation state of HCC. Restoring normal expression levels of these molecules could induce the tumor cells to differentiate into hepatocyte-like cells (HLCs) and suppress the malignant tumor phenotype. The strategies for inducing HLCs from induced pluripotent stem cells, fibroblasts, and other somatic cells provide a reference for the induced differentiation of liver cancer. The differentiation therapy is expected to be a promising and effective treatment for HCC.

Rapid ResaCeftazidime-Avibactam Enterobacterales NP Test: Rapid Detection of Ceftazidime-Avibactam Susceptibility in Enterobacterales.

Ceftazidime-avibactam (CZA), a novel ß-lactam/ß-lactamase inhibitor combination, has good antibacterial activity against carbapenem-resistant Enterobacterales (CRE) producing class A and C and some class D carbapenemases, but in recent years, the emergence of CZA-resistant Enterobacterales bacteria is growing. Therefore, rapid, accurate, and timely detection of CZA is necessary for clinical anti-infection treatment. In this study, the rapid ResaCeftazidime-avibactam Enterobacterales NP test was developed; its principle is that metabolically active bacteria (CZA-resistant strains) can change resazurin-PrestoBlue, a viability colorant, from blue to purple or pink in the presence of CZA, whereas CZA-susceptible strains cannot. We used 178 Enterobacterales isolates to evaluate the performance of this test. This test allowed the susceptibility of Enterobacterales to CZA to be detected within 4.5 h with an overall performance of 96% category agreement (CA), 7% major errors (MEs), and 0% very major errors (VMEs). Performance for Escherichia coli included 100% CA and 0% MEs and VMEs. Performance for Klebsiella pneumoniae included 99% CA and 2% MEs and 0% VMEs. Performance for Enterobacter cloacae included 87% CA, 25% MEs, and 0% VMEs. Moreover, this test is both economical ($1.0106 per isolate) and convenient, as it only requires basic laboratory equipment. In a word, the rapid ResaCeftazidime-avibactam Enterobacterales NP test is rapid and feasible, which may provide certain backing for the rapid screening and timely treatment of CZA-resistant strains in the clinic.

Cucurbitaceae genome evolution, gene function and molecular breeding.

The Cucurbitaceae is one of the most genetically diverse plant families in the world. Many of them are important vegetables or medicinal plants and are widely distributed worldwide. The rapid development of sequencing technologies and bioinformatic algorithms has enabled the generation of genome sequences of numerous important Cucurbitaceae species. This has greatly facilitated research on gene identification, genome evolution, genetic variation and molecular breeding of cucurbit crops. So far, genome sequences of 18 different cucurbit species belonging to tribes Benincaseae, Cucurbiteae, Sicyoeae, Momordiceae and Siraitieae have been deciphered. This review summarizes the genome sequence information, evolutionary relationship, and functional genes associated with important agronomic traits (e.g., fruit quality). The progress of molecular breeding in cucurbit crops and prospects for future applications of Cucurbitaceae genome information are also discussed.

Selenium Deficiency Aggravates Heat Stress Pneumonia in Chickens by Disrupting the M1/M2 Balance.

Selenium (Se) is an essential trace element found in the body. Se deficiency and M1/M2 imbalance are closely related to inflammation. Heat stress can decrease immune function and cause inflammation. In order to investigate whether Se deficiency can aggravate pneumonia caused by heat stress and the role of M1/M2 imbalance in the occurrence of pneumonia, 100 AA broilers were divided into two groups and fed the conventional diet (0.2 mg/kg Se) and the Se-deficient diet (0.03 mg/kg Se). After 40 days of feeding, the normal feeding group was randomly divided into a control group and a heat stress group. At the same time, the Se-deficient diet feeding group was randomly divided into a low Se group and a low Se heat stress group, with 25 chickens in each group. The model was established by exposure at 40℃. Six hours later, broilers were euthanized, and their lung tissues were collected. Hematoxylin and eosin staining, immunofluorescence, quantitative real-time PCR, and western blotting were used to detect lung histopathological changes and the expression of M1/M2 markers, nuclear receptor-κB (NF-κB) pathway genes, and heat shock proteins. Meanwhile, the activity and content of oxidative stress-related indices were also detected. We found that the expression of interleukin-1ß, interleukin-6, interleukin-12, and tumor necrosis factor-α was upregulated and the expression of interleukin-2, interleukin-10, and interferon-γ was downregulated. Immunofluorescence showed that the expression of CD16 was increased, the expression of CD163 was weakened, and the M1/M2 imbalance was present. In addition, the NF-κB pathway was activated by the increased expressions of heat shock proteins and oxidative stress. There was an increase in malondialdehyde, nitric oxide, and inducible nitric oxide synthase content, while the activity of total antioxidant capacity, glutathione peroxidase, catalase, and superoxide dismutase decreased, and the expression of NF-κB and cyclooxygenase-2 increased. These results suggest that low Se induces M1/M2 imbalance through oxidative stress activation of the NF-κB pathway and aggravates lung tissue inflammation caused by heat stress. This study offers a theoretical basis for exploring the pathogenesis of various kinds of inflammation induced by Se deficiency from the perspective of M1/M2 and provides a reference for the prevention of such diseases.

Revealing the Specific Regulations of Brassinolide on Tomato Fruit Chilling Injury by Integrated Multi-Omics.

Tomato fruit is susceptible to chilling injury (CI) when stored at low temperatures, limiting its storage potential, and resulting in economic loss if inappropriate temperatures are used. Brassinolide (BR) is a plant growth regulator that is known to decrease the susceptibility of fruit to CI. In this study, transcriptome, metabolome, and proteome analysis revealed the regulation mechanism of BR treatment in alleviating tomato fruit CI. The results showed that the differentially expressed metabolites mainly included amino acids, organic acids, carbohydrates, and lipids. Differentially expressed genes (DEGs) were involved in plant cold stress response (HSFA3, SHSP, and TPR), fruit redox process (POD, PAL, and LOX), related to the fruit texture (CESA, ß-Gal, and PAE), plant hormone signal transduction (ACS3, ARF, and ERF,), transcription factors (TCP, bHLH, GATA). Moreover, differentially expressed proteins were associated with fruit texture (CESA, PE, PL, and CHI), plant oxidation processes (LOX, GPX, CAT, and POD), plant cold stress response (HSF, HSP20, HSP70, and HSP90B), plant hormone signal transduction (BSK1 and JAR1) and transcription factors (WRKY and MYB). Our study showed that BR alleviates CI symptoms of tomato fruit by regulating LOX in the α-linolenic acid metabolism pathway, enhancing jasmonic acid-CoA (JA-CoA) synthesis, inhibiting cell wall and membrane lipid damage. The results provided a theoretical basis for further study on the CI mechanism of tomato fruit.

In vitro Activity of Contezolid Against Methicillin-Resistant Staphylococcus aureus, Vancomycin-Resistant Enterococcus, and Strains With Linezolid Resistance Genes From China.

Contezolid is a novel oxazolidinone, which exhibits potent activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and penicillin-resistant Streptococcus pneumoniae (PRSP). In this study, the in vitro activity of contezolid was compared with linezolid (LZD), tigecycline (TGC), teicoplanin (TEC), vancomycin (VA), daptomycin (DAP), and florfenicol (FFC) against MRSA and VRE strains isolated from China. Contezolid revealed considerable activity against MRSA and VRE isolates with MIC90 values of 0.5 and 1.0 µg/mL, respectively. For VRE strains with different resistance genotypes, including vanA- and vanM-type strains, contezolid did not exhibit significantly differential antibacterial activity. Furthermore, the antimicrobial activity of contezolid is similar to or slightly better than that of linezolid against MRSA and VRE strains. Subsequently, the activity of contezolid was tested against strains carrying linezolid resistance genes, including Staphylococcus capitis carrying cfr gene and Enterococcus faecalis carrying optrA gene. The results showed that contezolid exhibited similar antimicrobial efficacy to linezolid against strains with linezolid resistance genes. In general, contezolid may have potential benefits to treat the infections caused by MRSA and VRE pathogens.

Subacute cadmium exposure promotes M1 macrophage polarization through oxidative stress-evoked inflammatory response and induces porcine adrenal fibrosis.

Cadmium (Cd) is a widely distributed environmental pollutant with immunotoxicity and endocrine toxicity. M1/M2 macrophages participate in the immune response and exert an essential influence on fibrosis. Nevertheless, whether Cd can induce porcineadrenal fibrosis by affecting the polarization of M1/M2 macrophages and its potential regulatory mechanism have not been explored. We added 20 mg/kg CdCl2 to the pig diet for 40 days to investigate the fibrogenic effect of subacute Cd exposure on the adrenal gland. The results indicated that the ACTH and CORT in serum were decreased by 15.26 % and 21.99 %, respectively. The contents of adrenal mineral elements Cd, Cr, Mn were increased up to 34, 1.93, 1.42 folds and Co, Zn, Sn were reduced by 21.57 %, 20.52 %, 15.75 %. Concurrently, the pro-oxidative indicators (LPO, MDA and H2O2) were increased by 1.85, 2.20, 2.77 folds and 3.60, 11.15, 4.11 folds upregulated mRNA levels of TLR4, NF-κB, NLRP3 were observed. Subsequently, the expression of M1 macrophages polarization markers (IL-6, iNOS, TNF-α, CCL2 and CXCL9) were raised by 2.03, 2.30, 2.35, 1.58, 1.56 folds, while M2 macrophages (IL-4, CCL24, Arg1, IL-10, MRC1) showed a 62.34 %, 31.88 %, 50.26 %, 74.00 %, 69.34 % downregulation. The expression levels of AMPK subunits and genes related to glycolysis, oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) were also markedly increased. Additionally, the expression level of TGF-ß1, Smad2/3 and downstream pro-fibrotic markers was obviously upregulated. Taken together, we conclude that Cd activates the oxidative stress-mediated TLR4/NF-κB/NLRP3 inflammatory signal transduction, leading to porcine adrenal fibrosis by promoting macrophage polarization toward M1.

2-Methoxyestradiol synergizes with Erlotinib to suppress hepatocellular carcinoma by disrupting the PLAGL2-EGFR-HIF-1/2α signaling loop.

Erlotinib, an EGFR tyrosine kinase inhibitor has been introduced into cancer chemotherapy. However, the therapeutic effects of erlotinib in hepatocellular carcinoma (HCC) remain vaguely understood. Our previous study found that a hypoxia-mediated PLAGL2-EGFR-HIF-1/2α signaling loop in HCC decreased response to erlotinib. The current study has demonstrated that the combination of erlotinib and 2ME2 exerted synergistic antitumor effects against HCC. Further investigation showed that erlotinib increased the expression level of EGFR, HIF-2α, and PLAGL2, which contributes to the insensitivity of hypoxic HCC cells to erlotinib. The simultaneous exposure to 2ME2 effectively inhibited the expression level of EGFR, HIF-2α, and PLAGL2 that was induced by erlotinib. This contributes to the synergistic effect of the two therapeutic agents. Furthermore, the combination of erlotinib and 2ME2 induced apoptosis and inhibited the stemness of hypoxic HCC cells. Our findings potentially explain the mechanism of HCC insensitivity to erlotinib and provide a new strategy of combining EGFR and HIF1/2α inhibitors for HCC treatment.

Cadmium induces endoplasmic reticulum stress-mediated apoptosis in pig pancreas via the increase of Th1 cells.

Cadmium (Cd), an environmental pollutant, causes several adverse reactions in animals. High dose of Cd has serious cytotoxicities, including the induction of programmed cell necrosis, autophagy and apoptosis, which has aroused wide public concern. The balance of cytokine network is affected by Th1/Th2 balance which is closely related to immune response and the occurrence, development, treatment and outcome of various diseases. Cd can induce severe apoptosis, but the relationship between Cd induced apoptosis and Th1/Th2 balance has not been clarified. In this study, we established a pig Cd poisoning model, exposing to CdCl2 for 40 days (20 mg Cd/kg diet). Firstly, deviation of Th1/Th2 balance was observed by fluorescence staining, and apoptosis was observed by TUNEL staining. Then, real-time fluorescence quantitative analysis and Western blot were used to detect the expression of related proteins. The results show that Cd can interfere with the balance of Th1/Th2 and shift the balance towards Th1. In addition, through the experiments, we found that Cd exposure can increase the expression of glucose-regulated protein 94 (GRP94) and glucose-regulated protein 78 (GRP78), marker proteins of unfolded protein response (UPR). Cd exposure can increase the expression of pancreatic endoplasmic reticulum kinase (PERK), CCAAT-enhancer-binding protein homologous protein (CHOP), inositol-requiring enzyme 1 (IRE-1), activating transcription factor 6 (ATF-6), cysteinyl aspartate specific proteinase (Caspase12), indicating the three branches (ATF6, PERK and IRE-1) of endoplasmic reticulum stress (ER-stress) were activated. Moreover, we found that the expression of pro-apoptosis genes in the downstream pathway of ER-stress increased. In summary, our results indicated that Cd exposure upregulated the expression of pro-apoptosis related genes and caused apoptosis via the activation of the ER-stress signaling pathways in pancreas cells. And these negative effects were correlated with the equilibrium drift of Th1/Th2, increase in the expression and secretion of Th1 cytokines.

PLAGL2-EGFR-HIF-1/2α Signaling Loop Promotes HCC Progression and Erlotinib Insensitivity.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, hence a major public health threat. Pleomorphic adenoma gene like-2 (PLAGL2) has been reported to play a role in tumorigenesis. However, its precise function in HCC remains poorly understood. APPROACH AND RESULTS: In this study, we demonstrated that PLAGL2 was up-regulated in HCC compared with that of adjacent nontumorous tissues and also correlated with overall survival times. We further showed that PLAGL2 promoted HCC cell proliferation, migration, and invasion both in vitro and in vivo. PLAGL2 expression was positively correlated with epidermal growth factor receptor (EGFR) expression. Mechanistically, this study demonstrated that PLAGL2 functions as a transcriptional regulator of EGFR and promotes HCC cell proliferation, migration, and invasion through the EGFR-AKT pathway. Moreover, hypoxia was found to significantly induce high expression of PLAGL2, which promoted hypoxia inducible factor 1/2 alpha subunit (HIF1/2A) expression through EGFR. Therefore, this study demonstrated that a PLAGL2-EGFR-HIF1/2A signaling loop promotes HCC progression. More importantly, PLAGL2 expression reduced hepatoma cells' response to the anti-EGFR drug erlotinib. PLAGL2 knockdown enhanced the response to erlotinib. CONCLUSIONS: This study reveals the pivotal role of PLAGL2 in HCC cell proliferation, metastasis, and erlotinib insensitivity. This suggests that PLAGL2 can be a potential therapeutic target of HCC.

Integration of Moderation and Mediation in a Latent Variable Framework: A Comparison of Estimation Approaches for the Second-Stage Moderated Mediation Model.

An increasing number of studies have focused on models that integrate moderation and mediation. Four approaches can be used to test integrated mediation and moderation models: path analysis (PA), product indicator analysis (PI, constrained approach and unconstrained approach), and latent moderated structural equations (LMS). To the best of our knowledge, few studies have compared the performances of PA, PI, and LMS in evaluating integrated mediation and moderation models. As a result, it is difficult for applied researchers to choose an appropriate method in their data analysis. This study investigates the performance of different approaches in analyzing the models, using the second-stage moderated mediation model as a representative model to be evaluated. Four approaches with bootstrapped standard errors are compared under different conditions. Moreover, LMS with robust standard errors and Bayesian estimation of LMS and PA were also considered. Results indicated that LMS with robust standard errors is the superior evaluation method in all study settings. And PA estimates could be severely underestimated as they ignore measurement errors. Furthermore, it is found that the constrained PI and unconstrained PI only provide acceptable estimates when the multivariate normal distribution assumption is satisfied. The practical guidelines were also provided to illustrate the implementation of LMS. This study could help to extend the application of LMS in psychology and social science research.

Cadmium-induced oxidative stress promotes apoptosis and necrosis through the regulation of the miR-216a-PI3K/AKT axis in common carp lymphocytes and antagonized by selenium.

Cadmium (Cd) is a primary environmental pollutant which causes the immune dysfunction of aquatic animals. MicroRNAs (miRNAs) play a key role in programmed necrosis and apoptosis of immune organs. Selenium (Se), known as an important element, can antagonize Cd toxicity in birds, but the impact of Se on common carps (Cyprinus carpio) has not been reported. To investigate the Cd-induced immunotoxicity mechanism mediated by miR-216a in splenic lymphocytes of common carp and antagonized by Se, we extracted lymphocytes from the spleen and divided them into control group, Se group (10-6 mol/L of Na2SeO3), Se + Cd group and Cd group (4 × 10-5 mol/L of CdCl2). After 6 h of incubation, AO/EB staining, Flow cytometry, qPCR and Western blot were performed. The results showed that Cd exposure caused the apoptosis (BAX, Bcl-2, Caspase 3, Caspase 9) and programmed necrosis (RIP, RIP3, MLKL) in lymphocytes, increased the expression of CYP enzymes, glycometabolism-related enzymes and production of ROS, while irritated the oxidative stress (MDA, SOD, CAT and GSH-PX), upregulated the expression of miR-216a which attenuated the levels of PI3K. However, those variations were apparently mitigated in the Se + Cd group. In short, we have proven that Cd activates oxidative stress and miR-216a-PI3K/AKT axis disorder, thus promoting apoptosis and necrosis in lymphocytes. Moreover, Se can antagonize Cd-triggered apoptosis and necrosis in lymphocytes.

Selenium deficiency exacerbates LPS-induced necroptosis by regulating miR-16-5p targeting PI3K in chicken tracheal tissue.

Multiple tissue necrosis is one of the morphological features of selenium deficiency-mediated injury. MicroRNA (miRNA) participates in the occurrence and development of necroptosis by regulating target genes. Necroptosis is a programmed form of necrosis, and it is closely related to lipopolysaccharide (LPS)-induced injury. Our aim was to investigate whether Se deficiency can promote tracheal injury caused by LPS through miRNA-induced necroptosis. By establishing models of tracheal injury in Se-deficient chickens, we verified the targeting relationship between chicken-derived miR-16-5p and PI3K through bioinformatics, qRT-PCR and WB analyses, and we measured the changes in the expression of genes related to the PI3K/AKT pathway, RIP3/MLKL pathway and MAPK pathway and of heat shock proteins. Under the condition of Se deficiency, the following results were observed: PI3K/AKT expression decreased with the upregulation of miR-16-5p, the expression of necroptosis-related factors (TNF-α, RIP1, FADD, RIP3 and MLKL) increased, and the expression of Caspase 8 significantly decreased (p < 0.05). Light microscopy observations indicated that cell necrosis was the main pathological change due to Se deficiency injury in the tracheal epithelium. The MAPK pathway was activated, and HSP expression was upregulated, indicating that the MAPK pathway and HSPs are both involved in Se deficiency-mediated necroptosis. In addition, Se deficiency promoted the expression of necroptosis-related genes in LPS-treated chickens (p < 0.05), and the pathological changes of cell necrosis were more obvious. In conclusion, we demonstrated that Se deficiency regulates the miR-16-5p-PI3K/AKT pathway and exacerbates LPS-induced necroptosis in chicken tracheal epithelial cells by activating necroptosis-related genes.

Selenomethionine relieves inflammation in the chicken trachea caused by LPS though inhibiting the NF-κB pathway.

Selenomethionine is able to relieve the effect of inflammation in various tissues and organs. However, there are few studies about the influences of organic selenium resisting inflammation induced by LPS in chicken trachea. Therefore, the purpose of this experiment is to explore the organic selenium (selenomethionine) can raise immune function and relieve the LPS-induced inflammation of chicken trachea via inhibiting the NF-κB pathway. To investigate the mechanism of organic selenium on chicken trachea, the supplement of selenomethionine and/or LPS-induced chicken models were established. One hundred 46-week-old isa chickens were randomly divided into four groups (n = 25). The four groups were the control group, the selenomethionine group (Se group), the LPS-induced group (LPS group), and the Se and LPS interaction group (Se + LPS group). Then, the expressions of inflammatory factors (including induced nitric oxide synthase (iNOS), nuclear factor-kappa B(NF-κB), tumor necrosis factor (TNF-α), cyclooxygenase-2 (COX-2), and prostaglandin E (PTGEs) synthase), inflammation-related cytokines (including interleukin (IL-2, IL-6, IL-8, IL-17) and immunoglobulin (IgA, IgM, IgY)), the marker of immune function (avian ß-defensins (AvBD6, AvBD7)), heat shock proteins (including HSP60, HSP90), and selenoproteins (including Selo, Sels, Selm, Selh, Selu, Seli, SPS2, GPx1, GPx2, Dio1, Sepx1, Sep15, Sepp1, Txnrd1) were detected in our experiment. The above genes were significantly changed in different groups (p < 0.05). We can conclude that organic selenium can increase the function of immunity and the expression of selenoproteins, and mitigate the inflammation induced by LPS via suppression of the NF-κB pathway.

Avermectin inhibits neutrophil extracellular traps release by activating PTEN demethylation to negatively regulate the PI3K-ERK pathway and reducing respiratory burst in carp.

Excessive residual avermectin (AVM) in the environment can have toxic effects on non-target organisms. AVM can exert immunotoxicity by inducing genomic demethylation, but its effect on neutrophil extracellular traps (NETs) release in carp is unclear. In this study, carp neutrophils were pretreated with 5 µg/L AVM or 4 µM DNA demethylation inhibitor (aurintricarboxylic acid, ATA), alone or in combination, and then treated with 4 µM phorbol 12-myristate 13-acetate (PMA) to stimulate NETs release. The results showed that exposure of carp neutrophils to AVM significantly suppressed NETs release and MPO expression, increased ROS production, and dramatically reduced PMA-induced cellular respiratory burst. In addition, AVM could bind to the MBD2 molecule, markedly upregulate MBD2 expression to cause demethylation, and clearly activate PTEN expression, thereby inhibiting the expression of PI3K, AKT, Raf, MEK, and ERK. However, these effects were alleviated by ATA. In conclusion, our study showed that AVM could inhibit NETs release in carp by inducing demethylation of PTEN to negatively regulate NETs synthesis pathways and reducing respiratory burst level. Our findings clarify the mechanism of AVM immunotoxicity to fish and are of great significance for efforts to protect the ecological environment and human health.

Chlorpyrifos induced oxidative stress to promote apoptosis and autophagy through the regulation of miR-19a-AMPK axis in common carp.

Chlorpyrifos (CPF) has become a mainly pollution in water environment. Micro-RNAs (miRNAs) play an important part in the development of apoptosis and autophagy. However, the potential mechanism of CPF induced kidney toxicity and the roles of miRNAs are still unclear. To explore the underlying mechanism, the kidney of common carp exposed to different concentrations of CPF for 40 days was used as a research object. We found that CPF could damage the ultrastructure and function of kidney; and also caused antioxidant system disorder. CPF inhibited the mRNA level of miR-19a which improved AMP-activated protein kinase (AMPK). Furthermore, the detection of apoptosis and autophagy relative genes showed that the expressions of TSC complex subunit 2 (TSC2), light chain 3 (LC3), Dynein, tumor protein 53 (p53), Bcl-2 associated X protein (Bax), caspase-3 and caspase-9 were enhanced and the expressions of nechanistic target of rapamycin (mTOR), Ras homolog mTORC1 binding (Rheb) and B-cell lymphoma (Bcl-2) were reduced in dose-dependent way. Taken together, we conclude that CPF causes oxidative stress and miR-19a-AMPK axis disorder, thereby promotes apoptosis and autophagy in common carp kidney. Our study will provide theoretical basis for toxicology research and environmental protection of CPF.

Chlorpyrifos Suppresses Neutrophil Extracellular Traps in Carp by Promoting Necroptosis and Inhibiting Respiratory Burst Caused by the PKC/MAPK Pathway.

Neutrophil extracellular traps (NETs) are reticular structures formed by myeloperoxidase (MPO), histones, and neutrophil elastase (NE) that are released from neutrophils in response to pathogenic stimuli. Chlorpyrifos (CPF) is wildly used as an organophosphorus pesticide that causes a range of toxicological and environmental problems. Exposure to CPF can increase the production of neutrophils in carps, and this increase can be considered a biomarker of water pollution. To explore a relationship between NETs and CPF and its mechanism of influence, we treated neutrophils from the blood of carp with 1 µg/mL phorbol 12-myristate 13-acetate (PMA), 0.325 mg/L CPF, or 20 µM necrostatin-1 (Nec-1). The production of MPO and NETs was reduced in the CPF+PMA group compared with that in the PMA group. CPF can cause an increase in reactive oxygen species (ROS), while inhibiting respiratory burst caused by PMA stimulation. We found that the expression levels of protein-coupled receptor 84 (gpr84), dystroglycan (DAG), proto-oncogene serine/threonine kinase (RAF), protein kinase C (PKC), and mitogen-activated protein kinase 3 (MAPK3) in the CPF+PMA group were lower than those in the PMA group, indicating that the PKC-MAPK pathway was suppressed. The expression levels of cylindromatosis (CYLD), mixed lineage kinase domain-like pseudokinase (MLKL), receptor-interacting serine-threonine kinase 1 (RIP1), and receptor-interacting serine-threonine kinase 3 (RIP3) were increased, and the expression levels of caspase 8 were reduced by CPF, indicating that CPF may cause necroptosis. The addition of Nec-1 restored the number of NETs in the CPF+PMA group. The results indicate that CPF reduced the production of NETs by inhibiting respiratory burst and increasing necroptosis. The results contribute to the understanding of the immunotoxicological mechanism of CPF and provide a reference for comparative medical studies.

Oxidative stress, inflammation, and glycometabolism disorder-induced erythrocyte hemolysis in selenium-deficient exudative diathesis broilers.

Selenium (Se) deficiency causes injury of diversified tissues and cells, including livers, hearts, skeletal muscles, and erythrocytes. The aim of the present study is to explore the molecular mechanism of erythrocyte hemolysis due to Se deficiency in broilers. One hundred and eighty broilers (male/female, 1 day old) were randomly divided into two groups and fed with either a normal Se content diet (C group, 0.2 mg Se/kg) or a Se-deficient diet (ED group, 0.008 mg Se/kg) for 45 days. During the trial period of 15-30 days, biological properties such as osmotic fragility, fluidity, phospholipid components of cell membrane, adenosine triphosphatase activities, and antioxidant function of erythrocytes in broilers were examined. Moreover, the messenger RNA (mRNA) expressions of genes associated with inflammation, glycometabolism, and avian uncoupling protein (avUCP) were detected. We found that compared with the C group, hemolysis rate, degree of polarization, and microviscosity of erythrocytes were increased in broilers of the ED group. The composition of erythrocyte membrane lipids was changed. Meanwhile, the antioxidant function of erythrocytes was weakened and mRNA levels of inflammatory genes were stimulated by Se deficiency (p < 0.05). In addition, mRNA expressions of rate-limiting enzymes in glycometabolism were effected and avUCP mRNA level was downregulated (p < 0.05) in the ED group. It has been concluded from the results that oxidative stress, inflammatory response, and glycometabolism disorder lead to erythrocyte hemolysis by changing the structure and function of erythrocyte membrane in ED broilers suffered from Se deficiency.

The Effects of Low Selenium on DNA Methylation in the Tissues of Chickens.

DNA methylation is involved in epigenetic mechanisms associated with gene suppression, and its abnormalities lead to gene instability and disease development. As an essential trace element in humans and animals, selenium (Se) is also associated with abnormal changes in DNA methylation. However, the effect of low Se on DNA methylation in avian tissues has not been reported. In the current study, chickens were fed a low-Se diet (0.033 mg Se/kg) or supplemented with 0.15 mg Se/kg as selenite for up to 55 days. DNA methylation levels were examined by high-performance liquid chromatography (HPLC). DNA methyltransferases (DNMTs) and methyl-DpG-binding domain protein 2 (MBD2) mRNA levels were examined through the applications of RT-PCR. The experiment aims to explore the relationship between low Se and DNA methylation. The results showed that total DNA methylation levels in the muscle tissues, brain, immune tissues, and liver of the low-selenium diet group were decreased compared with the control group. The degree of DNA methylation reduction in different tissues from largest to smallest was liver > cerebellum > thymus > brain > spleen ≥ leg muscles > pectoral muscles > bursa of Fabricius > thalamus > wing muscles. DNMT1, DNMT3A, and DNMT3B mRNA expression levels of the low-selenium diet group were decreased compared with those in the control group. The mRNA expression of the MBD2 gene was increased. The results indicate that low Se can reduce the DNA methylation levels of tissues, especially within the liver. These conclusions provide a basis for exploring the pathogenesis of selenium deficiency from the perspective of DNA methylation and create a new basis for comparative medicine.