The toxic effects induced by benzethonium chloride on Daphnia carinata over two generations: Resistance and higher sublethal toxicity.

With the widespread utilization of the sanitizing product benzethonium chloride (BEC) throughout the coronavirus pandemic, concerns have emerged regarding its potential hazards. Nevertheless, the long-term and multigenerational toxic effects of BEC on aquatic organisms remains unexplored. This study investigates acute and chronic toxicity, oxidative stress, mitochondrial membrane potential, ATP concentrations, and gene expression using Daphnia carinata as the model organism. Meanwhile, hierarchical clustering analysis was utilized to investigate phenotypic effects among different treatment groups. The integrated biomarker response index version 2 (IBRv2) was employed to estimate the deviation in toxic effects over two generations. These results indicated that D. carinata in the second generation exhibited higher survival rate and lower levels of oxidative stress than the first generation. However, the higher sublethal effects were found in the second generation as follows, the weakened growth performance, mitochondrial membrane potential depolarization, reduced ATP concentrations, and down-regulated gene expression. The mitochondrial toxicity induced by BEC may account for the distinct toxic effects exhibited in two generations. The findings here can assist with the evaluation of potential risk for BEC on aquatic organisms, and provide new insight into the cross-generational toxicity mechanisms of pollutants in aquatic ecosystems.

NF-κB Inhibitor Myrislignan Induces Ferroptosis of Glioblastoma Cells via Regulating Epithelial-Mesenchymal Transformation in a Slug-Dependent Manner.

Glioblastoma (GBM) is the most common malignant tumor of the adult central nervous system. Aberrant regulation of cell death is an important feature of GBM, and investigating the regulatory mechanisms of cell death in GBM may provide insights into development of new therapeutic strategies. We demonstrated that myrislignan has ferroptosis-promoting activity. Myrislignan is a lignan isolated from Myristica fragrans Houtt and an inhibitor of NF-κB signaling pathway. Ferroptosis is an iron-dependent form of programmed cell death characterized by the accumulation of intracellular lipid peroxidation products. Interestingly, ferroptosis was associated with other biological processes in tumor cells such as autophagy and necroptosis. Recently, the crosstalk between epithelial-mesenchymal transition (EMT) and ferroptosis has also been reported, but the mechanisms underlying the crosstalk have not been identified. Our results indicated that myrislignan suppressed growth of GBM through EMT-mediated ferroptosis in a Slug-dependent manner. Myrislignan inhibited the activation of NF-κB signaling by blocking the phosphorylation of p65 protein and induced ferroptosis through the Slug-SLC7A11 signaling pathway in GBM cells. In addition, myrislignan suppressed the progression of GBM in xenograft mouse model. Hence, our findings contribute to the understanding of EMT-induced ferroptosis and provide targets for the development of targeted therapy against GBM.

Transcriptome profiles of red swamp crayfish Procambarus clarkii hematopoietic tissue in response to WSSV challenge.

The crayfish Procambarus clarkii could achieve a high cumulative mortality after WSSV infections. To better understand the immune response to WSSV in hematopoietic tissue, the present study investigated the immunological response of P. clarkii and analyzed the expression of some hematopoietic cytokines. After assembly, there was an average of 47,712,411 clean reads were obtained in control and treatment groups. A total of 35,945 unigenes were discovered with N50 length of 1554 bp. Under functional classification, enrichment, and pathway analysis using different database, there were about 257 differentially expressed genes (DEGs) identified, of which 139 were up-regulated and 118 were down-regulated. The GO function analysis of these DEGs were mostly participated in activation of immune response, complement activation, complement binding, negative regulation of humoral immune response and secretory granule membrane. Under KEGG analysis, these DEGs were involved in ECM-receptor interaction, HIF-1 signaling pathway, Glycolysis/Gluconeogenesis, Thyroid hormone signaling pathway and Glucagon signaling pathway. The real-time quantitative PCR (RT-qPCR) analysis of 9 selected genes confirmed the reliability of RNA-Seq results. The present research provide for the first time the transcriptomic profile of P. clarkii hematopoietic tissue in response to WSSV infection and reveals the astakines may play important roles in antiviral immune response. The results of the present study will further enrich the theoretical basis of the crayfish immune system and provide new ideas for disease prevention and control.

Bisphenol A disrupts apolipoprotein E expression through estrogen-related receptor gamma and DNA methlylation in the liver of male rare minnow Gobiocypris rarus.

An increasing number of studies show that bisphenol A (BPA) can cause lipid metabolism disorder. However, few studies focused on the effect of BPA on lipid transport. Apolipoprotein E (ApoE) plays important roles in triglyceride (TG) transportation. Our previous study found that ApoE was a sensitive gene in response to BPA exposure in male rare minnow. To investigate the effect and mechanism of BPA on hepatic ApoE, adult male rare minnow Gobiocypris rarus were exposed to environmentally relevant concentrations of BPA (15 µg/L) for 1, 3 and 5 weeks. Results showed that BPA inhibited ApoE expression at week 1 and 5, while induced its expression at week 3. A positive estrogen-related receptor gamma (Esrrg) response element was identified in the promoter region of ApoE. The change of the Esrrg recruitment was consistent with ApoE mRNA expression. Moreover, the methylation status of the CpG sites near and on the Esrrg binding sites changed opposite to the ApoE mRNA level, which may be the main cause for the change in Esrrg recruitment. The expression of ApoE protein was significantly enhanced following long-term BPA exposure. Consistently, the TG accumulation was significantly increased in the plasma. The present study demonstrates that BPA could affect rare minnow ApoE expression, which is probably one of the ways for BPA disturbing fish lipid metabolism.

Bisphenol A regulates apolipoprotein A1 expression through estrogen receptors and DNA methlylation and leads to cholesterol disorder in rare minnow testis.

Bisphenol A (BPA) is a well-known plasticizer that widely distributed in the aquatic environment. BPA has many adverse effects on reproduction. However, few studies have investigated the mechanism of BPA affecting reproduction from the perspective of lipid metabolism. Apolipoprotein A1 (ApoA1) is the major component of high-density lipoprotein (HDL), and plays critical roles in reverse cholesterol transport (RCT). In this study, in order to investigate the effect and molecular mechanism of BPA on testicular ApoA1 and the role of ApoA1 in BPA induced abnormal spermatogenesis, adult male rare minnow Gobiocypris rarus were exposed to 15 µg/L of BPA for 1, 3 and 5 weeks. Results showed that BPA could significantly affect testicular ApoA1 mRNA and protein levels, testicular cholesterol levels, plasmatic sex hormone levels and the integrity of sperm head membrane. The main mechanism of BPA regulating ApoA1 expression is to alter Esr recruitment and CpG sites DNA methylation in ApoA1 promoter. The induced ApoA1 up-regulated high density lipoprotein cholesterol levels and enhanced RCT, and finally decreased the testicular free cholesterol levels. This is likely a key mechanism by which BPA induces sex hormone disorder and sperm head membrane damage. The present study reveals the mechanism by which BPA interferes with spermatogenesis from the perspective of cholesterol transport.

The identification of a nuclear factor Akirin with regulating the expression of antimicrobial peptides in red swamp crayfish (Procambarus clarkii).

Akirin is a highly conserved nuclear factor among different species. It is closely related to skeletal muscle development, innate immune response, and tumorigenesis in a variety of animals. In invertebrates, Akirin is mainly involved in gene transcription and NF-κB dependent natural immune response. In the present study, a nuclear factor Akirin was identified from Procambarus clarkii. The Akirin protein of crayfish consists of 204 amino acids and is conserved among its family members, especially the nuclear localization signal peptide motif (KRRR). PcAkirin was highly expressed in stomach, intestines, and hepatopancreas. After A. hydrophila challenge, the transcription level of Akirin significantly increased in hemocyte and hepatopancreas. In addition, the recombinant Akirin protein was produced successfully and helpful to resist WSSV infection by increasing the expression level of some immune related genes. On the contrary, after interfering with Akirin gene by dsRNA, the crayfish increased the sensitivity to A. hydrophila and WSSV infections. The results are more obvious in the accumulated mortality of P. clarkii infected with A. hydrophila and WSSV. All these results suggested that Akirin played a significant role in innate immune responses and protected it from WSSV and bacterial infection in crayfish.

A trypsin-like serine protease domain of masquerade gene in crayfish Procambarus clarkii could activate prophenoloxidase and inhibit bacterial growth.

Masquerade (Mas) is a secreted trypsin-like serine protease (SPs) and involved in immune response in some arthropods. However, according to previous studies, Mas presents different functional activities. In the present study, the functional mechanisms of Mas in crayfish Procambarus clarkii immune defense were studied. A fragment cDNA sequence of PcMas was identified and characterized. From the structural analysis, it contains a trypsin-like serine protease domain. The highest expression level of PcMas was detected in hepatopancreas. The infection of A. hydrophila could induce the expression of PcMas, while the WSSV infection did not cause changes in the expression of PcMas. Through the prokaryotic expression system, the PcMas protein was expressed in E. coli. It was verified that PcMas can bind to bacteria in vitro and inhibit the growth of the bacteria. By dsRNA interference with the expression of PcMas, the decrease expression of PcMas led to a decrease in the activity of phenoloxidase in hemolymph and an increase of mortality caused by A. hydrophila infection. The injection of recombinant protein can enhance the activity of phenoloxidase and reduce mortality caused by A. hydrophila infections. Therefore, the present study confirmed that PcMas could improve the body's immune response to eliminate bacterial pathogens by binding with bacteria and activating the prophenoloxidase system. The results will enrich the molecular mechanisms of crustaceans immune defense.

Acetochlor affects zebrafish ovarian development by producing estrogen effects and inducing oxidative stress.

Acetochlor is one of the most widely used pesticides worldwide and widely distributed in the water environment. However, studies on the reproductive influence of acetochlor are still limited. To investigate the impact and potential mechanism of acetochlor on fish ovarian development, zebrafish were utilized as experiment models. The ovarian histology, ovarian development-related genes, and plasma oxidative stress-related indexes were investigated following acetochlor (at nominal concentration 1, 10, and 100 µg/L) exposure for 7 and 21 days. Results showed that low-dose acetochlor had estrogen effect and induced zebrafish estradiol (E2) and ovarian vitellogenin (Vtg) synthesis and promoted ovarian development, while long-term exposure to higher doses of acetochlor reduced the ability of ovarian resistance to oxidative stress and destroyed the development of the ovary. Moreover, bone morphogenetic protein 15 (bmp15) and growth differentiation factor 9 (gdf9) were also involved in the influence of acetochlor on the ovarian development of zebrafish.

Characterization of a classical 2-cysteine peroxiredoxin1 gene from Chinese soft-shelled turtle Pelodiscus sinensis with its potent antioxidant properties and putative immune response.

Peroxiredoxin family members could function in host defense against oxidative stress, and modulate immune response. In the present study, a 2-cysteine peroxiredoxin gene named PsPrx1 was isolated from Chinese soft-shelled turtle Pelodiscus sinensis. The PsPrx1 cDNA was composed of 1130 bp, consisted of 199 amino acid residues and included a Redoxin and AphC-TSA domain. As detected by qPCR, PsPrx1 was ubiquitously expressed in the examined tissues with the higher levels in liver and spleen. Upon the immune challenge with A. jandaei bacteria and oxidative stress with ammonia pressure, both mRNA and protein expression level in liver could be significantly enhanced. The results of immunohistochemical examinations showed PsPrx1 was mainly distributed at the junction between the hepatic cells. The general functional properties of PsPrx1 were confirmed using purified rPsPrx1 protein. From the results, rPsPrx1 protein was confirmed to exhibit antioxidant activity and antibacterial properties. The potential for scavenging extracellular H2O2 was evidenced by the purified rPsPrx1 protein in vitro system. In the mixed-function oxidase assay, rPsPrx1 also exhibited a dose-dependent inhibition of DNA damage. These results suggest that rPsPrx1 was implicated defense against microbial pathogens and oxidants, and would provide important information to further understand the functional mechanism of Prx1 in P. sinensis immunity.

MDA5 and LGP2 acts as a key regulator though activating NF-κB and IRF3 in RLRs signaling of mandarinfish.

RIG-I-like receptors (RLRs), as key cytoplasmic sensors of viral pathogen-associated molecular patterns, can recognise viral RNA and enhance the antiviral response. Some investigations have focused on the roles of RLRs in the innate immune response in grass carp, large yellow croaker, and rainbow trout. However, little is known about the function of RLRs in mandarinfish (Siniperca chuatsi), an important economic fish in Perciformes. Here, we functionally characterized the RLRs involved in the immune responses of mandarinfish (Siniperca chuatsi), by evaluating three RLRs, namely, RIG-I, MDA5, and LGP2. The results revealed that MDA5 and LGP2 were present in mandarinfish, whereas RIG-I was absent. The MDA5 and LGP2 cDNA sequences contained 2976 and 2046 bp and encoded 991 and 681 amino acids, respectively. Multiple sequence alignments showed that MDA5 and LGP2 of mandarinfish were clustered together with their homologs from other teleost fishes and shared high similarities with those from other vertebrates, and RIG-I of mandarinfish was absent. Moreover, quantitative real-time PCR (qPCR) analysis suggested that MDA5 and LGP2 were constitutively expressed in all tissues tested, and MDA5 mRNA expression was relatively high in the gill, and spleen, whereas LGP2 mRNA expression was high in the liver, gill, and head kidney. After stimulation with lipopolysaccharide or poly I:C, the expression of MDA5 and LGP2 was upregulated in spleen, gill and head kidney, but the pattern was not exactly the same, MDA5 transcripts generally increased and then declined with the prolonged infection, while LGP2 transcripts went up continuously, which showed that mandarinfish MDA5 and LGP2 may play independent roles in antiviral response. Besides, it is further revealed that the MDA5 could activate NF-κB and IRF3 to inducing the production of IFN-ß by constructing tet-on stable strain of 293T cell, however over-expression of LGP2 resulted in decreased NF-κB, IRF3 and IFN-ß production in cells challenged with LPS and polyI:C Taken together, our results demonstrated that MDA5 and LGP2, as a positive and negative regulator, respectively, played an important role in modulating antibacterial andantiviral immune responses though activating NF-κB and IRF3 in RLRs signaling of mandarinfish.

[Establishment of a transgenic cell model for preliminary screening of chemopreventive agents].

To develop a GFP transgenic cell model under the transcriptional control of TK promoter adjacent to which ARE enhancer was inserted. Synthetic oligonucleotide ARE motif was annealed and purified then inserted into pTK-GFP to construct the vector of pARE-TK-GFP. The TK and ARE-TK fragments were amplified by PCR and cloned into pEGFP-N1 to reconstruct eukaryotic expression vectors of pTK-GFP/Neo and pARE-TK-GFP/Neo. They were transfected into HepG2 cells and clones resistant G418 were isolated. PDTC and Lentinan were used to induce the cell levels of GFP and the fluorescence was measured using a fluorescence plate reader. The results showed that the induced level of GFP is significantly increased and have dose-dependeny in a certain range. This findings indicated that such a cell model offered a potential platform for preliminary screening of all kinds of natural or synthetic chemopreventive agents.