Suppressor of cytokine signaling 3 (SOCS3) is related to pro-inflammatory cytokine production and triglyceride deposition in turbot (Scophthalmus maximus).

Turbot (Scophthalmus maximus) is an economically important fish that is farmed by aquaculture for human consumption. Aquacultured turbot are commonly fed a high-lipid diet; however, this diet causes excessive lipid deposition and the overexpression of pro-inflammatory cytokines. Studies in mammals have indicated that a relationship exists between pro-inflammatory cytokine overexpression and altered lipid metabolism through the activation of suppressor of cytokine signaling 3 (SOCS3). In this study, we investigated the relationship between SOCS3 and triglyceride (TG) deposition and mechanism of SOCS3 activation in farmed turbot fed high-lipid diet (HLD). TG content increased with SOCS3 production, mediated by toll-like receptor-nuclear transcription factor kappa-B (TLR-NFκB) signaling in the liver of turbot fed a HLD and in turbot primary liver cells incubated with oleic acid (OA). Overexpression of SOCS3 increased TG deposition via the increased production of mature sterol regulatory element binding protein 1 (m-SREBP-1). Knockdown of SOCS3 in turbot primary liver cells resulted in normalized TG deposition and decreased m-SREBP-1 production. These results suggest that the HLD and OA can induce cytokine expression by activating the TLR-NFκB signaling pathways, resulting in increased SOCS3 expression. It is proposed that SOCS3 enhances m-SREBP-1 production, leading to TG deposition. These findings provide important new insights into the relationship between cytokine expression and TG deposition and mechanism of HLD-induced pro-inflammatory response, which could help to improve the health of farmed turbot and a better understanding of fish immunity.

Telomerase Reverse Transcriptase Gene from Greasyback Shrimp Metapenaeus ensis: Isolation, Genomic Organization and Expression Pattern Analysis.

Telomerase reverse transcriptase (TERT), a catalytic subunit of telomerase, plays a key role in the activity and biological functions of telomerase. In the present study we isolated and characterized the full-length cDNA and DNA sequences of the TERT gene (MeTERT) from Metapenaeus ensis. MeTERT cDNA was 4239 bp in length, which consisted of a 369 bp 5'UTR, a 3231 bp open reading frame encoding 1076 amino acids, and a 639 bp 3'UTR. The genomic DNA of MeTERT had only two introns, similar to beetle (two introns) and silkworm (intronless). The MeTERT protein showed only 5.2-7.9% identity with other known TERTs but contained all the four primary TERT domains of the N-terminal TEN, RNA binding domain (TRBD), reverse transcriptase (RT) and C-terminus CTE. Expression pattern analysis by RT-qPCR showed that, the MeTERT mRNA transcripts could be detected in all the tested samples, with relatively higher expression level in the gill, mysis, Oka organ and egg, but lower level in muscle, ovary, in vitro cultured 3-d Oka organ cells and heart. The significant decrease of MeTERT expression in the in vitro cultured 3-d Oka organ primary cells compared with their source tissue of Oka organ may have contributed to the cellular mitosisarrest. Thus trans-activation of TERT gene may be a candidate in attempts to immortalize in vitro cultured shrimp cells. This work will lay a solid foundation for future studies of the biological functions of telomerase in crustaceans.

A novel medium for long-term primary culture of hemocytes of Metapenaeus ensis.

The development of a suitable shrimp cell medium is essential for achieving a long-term cell culture and finite cell line of shrimps routinely. In this study, we have successfully developed an optimal shrimp cell medium that can be used for long-term in vitro culture and continuous subculture of the hemolymph cells (or hemocytes) of greasyback shrimp Metapenaeus ensis, designated as MeH cells, by shrimp serum-based and supplements-based optimization of the basic and growth medium. In this article, we have focused on the details for the preparation of the optimal shrimp cell medium by diluting and mixing of various stock solutions as well as the methods for isolation and primary culture of MeH cells.•A novel shrimp cell growth medium is developed for long-term shrimp hemocytes culture.•The preparation method of shrimp cell growth medium is successfully established.•Obvious cell activity and proliferation potential of isolated shrimp cells can be maintained beyond 30 days.

Auxenochlorella pyrenoidosa extract supplementation replacing fetal bovine serum for Carassius auratus muscle cell culture under low-serum conditions.

Cultured meat production requires large-scale cell proliferation in vitro with the supplementation of necessary media especially serum. This study investigated the capacity of Auxenochlorella pyrenoidosa extract (APE) to replace fetal bovine serum (FBS) for cell culture under low-serum conditions using Carassius auratus muscle (CAM) cells. Supplementation with APE and 5% FBS in the culture media significantly promoted the proliferation of CAM cells and increased the expression of MyoD in cells compared to that with 5% FBS through cell counting kit-8 and immunofluorescence staining assay. In addition, CAM cells in the media containing 5% FBS and APE could be continually cultured for 4 passages, and the cell number was 1.58 times higher than the counterpart without APE in long-term culture. Moreover, supplementation with APE realized large-scale culture on microcarriers under low-serum conditions, and more adherent cells were observed on microcarriers in 2% FBS supplemented with APE, compared with those in 2% FBS and 10% FBS without APE. These findings highlighted a potentially promising application of APE in muscle cell culture under low-serum conditions for cultured meat production.

Ring-stacked capsids of white spot syndrome virus and structural transitions with genome ejection.

White spot syndrome virus (WSSV) is one of the largest DNA viruses and the major pathogen responsible for white spot syndrome in crustaceans. The WSSV capsid is critical for genome encapsulation and ejection and exhibits the rod-shaped and oval-shaped structures during the viral life cycle. However, the detailed architecture of the capsid and the structural transition mechanism remain unclear. Here, using cryo-electron microscopy (cryo-EM), we obtained a cryo-EM model of the rod-shaped WSSV capsid and were able to characterize its ring-stacked assembly mechanism. Furthermore, we identified an oval-shaped WSSV capsid from intact WSSV virions and analyzed the structural transition mechanism from the oval-shaped to rod-shaped capsids induced by high salinity. These transitions, which decrease internal capsid pressure, always accompany DNA release and mostly eliminate the infection of the host cells. Our results demonstrate an unusual assembly mechanism of the WSSV capsid and offer structural insights into the pressure-driven genome release.

Establishment, characterization, and transfection potential of a new continuous fish cell line (CAM) derived from the muscle tissue of grass goldfish (Carassius auratus).

A new continuous fish cell line (CAM) has been successfully derived from the muscle tissues of grass goldfish, Carassius auratus. The primary cell cultures were initiated by incomplete trypsinization first and then explant culture in a Leibovitz-15 medium supplemented with 15% fetal bovine serum and 10% fish muscle extract. It was found that the CAM cells were very sensitive to trypsinization and needed to be sub-cultured at a low trypsin concentration of 0.0625% to be able to go through the crisis of spontaneous immortalization transformation, and afterward a total of five derivative cell strains were isolated from the original CAM cell line. This spontaneous immortalization transformation event was recorded successively at passages 44-47, beginning with a large-scale apoptosis and senescence and followed by mitosis arrest and re-activation, thus designated as cell strain CAM-44A, 44B, 45A, 44B, and 47A. Now both the CAM cell line and strains had been sub-cultured for more than 89 times and could be well cryopreserved in the growth medium containing 5% dimethylsulfoxide. Chromosome analysis and COI gene analysis had confirmed the grass goldfish origin of these CAM cells. Transfection potential analysis indicated that Lipofectamine LTX and Xfect were two suitable transfection reagents to be used in the gene delivery of CAM cells with a transfection efficiencies up to 11±6% and 8±3% in the CAM cell lines, respectively. Among the five cell strains, CAM-47A showed the highest transfection potential with a transfection efficiency up to 28 ± 5%. This work will provide a useful cell source for works on the cell-based artificial fish meat production and functional studies of fish myogenesis-related genes.

The Improvement and Application of Lentivirus-Mediated Gene Transfer and Expression System in Penaeid Shrimp Cells.

This study first reported the improvement and application of lentivirus-mediated gene transfer and expression system in shrimp cells. After modified by the inclusion of two envelope proteins (VP19 and VP28) of shrimp white spot syndrome virus (WSSV) into the envelope of the packaged lentivirus, and insertion of a truncated promoter of immediate-early gene 1 (Pie1-504) of shrimp WSSV virus into the lentiviral reporter plasmid, the second-generation lentiviral expression system (pLVX-PEF1α-IRES-mCherry, psPAX2, and PMD2.G) was found to behave better in the mitosis-arrested shrimp cells than the similarly modified retrovirus expression system did. Results from the insect sf9 cells indicated that the inclusion of VP19 and VP28 into the envelope of packaged lentiviruses could significantly improve the tropism or infectivity of the modified lentiviruses to insect cells in a cumulative way. Notably, the VP28 contributed about 86% of the total increase of the tropism. In the shrimp primary lymphoid cells infected by modified lentivirus IV with both VP19 and VP28 included, the infection efficiency was up to 11% (non-confocal) and 19% (confocal) and no background fluorescent signal was observed. However, background fluorescent signal was observed in the shrimp primary Oka organ cells although only under a confocal microscope. In the lentivirus IV-infected Oka organ cells, the actual infection efficiencies were calculated up to 8% (non-confocal) and 19% (confocal), significantly higher than those of commercial intact lentivirus I of 0 (non-confocal) and 3% (confocal). The insertion of WSSV promoter (Pie1-504) had interrupted the effective expression of reporter plasmid encoding lentiviral construct of pLVX-PEF1α-Pie1-504-IRES-mCherry in the HEK293T cells, but markedly increased its efficiencies up to 14% (non-confocal) and 26% (confocal) in the Oka organ cells. This improved lentivirus expression system will provide us a useful tool for efficient gene transfer and expression in shrimp cells.

Nonylphenol Causes Decrease in Antioxidant Enzyme Activities, Increase in O(2)(-) Content, and Alteration in Ultrastructures of FG Cells, a Flounder (Paralichthys olivaceus) Gill Cell Line.

ABSTRACT The findings in this study were that the exposure of FG cells to nonylphenol (NP) caused decreases in the cell growth rate, antioxidant enzyme activities, and ATP content; an increase in intracellular O(2)(-) content; and alteration of the organelles such as mitochondria and the RER. It is proposed that the reduction in antioxidant enzyme activities leads to accumulation of O(2)(-) content, which in turn results in the damage of mitochondrial infrastructures and a decline in energy production, which then causes the ultrastructural alterations in the cells. In addition, we show that FG cells are a good candidate system for evaluation of NP acute toxicities.

Reformation of tissue balls from tentacle explants of coral Goniopora lobata: self-organization process and response to environmental stresses.

Coral has strong regeneration ability, which has been applied for coral production and biodiversity protection via tissue ball (TB) culture. However, the architecture, morphological processes, and effects of environmental factors on TB formation have not been well investigated. In this study, we first observed TB formation from the cutting tentacle of scleractinia coral Goniopora lobata and uncovered its inner organization and architecture by confocal microscopy. We then found that the cutting tentacle TB could self-organize and reform a solid TB (sTB) in the culture media. Using chemical drug treatment and dissection manipulation approaches, we demonstrated that the mechanical forces for bending and rounding of the cutting fragments came from the epithelial cells, and the cilia of epithelial cell played indispensable roles for the rounding process. Environmental stress experiments showed that high temperature, not CO2-induced acidification, affected TB and sTB formation. However, the combination of high temperature and acidification caused additional severe effects on sTB reformation. Our studies indicate that coral TB has strong regeneration ability and therefore could serve as a new model to further explore the molecular mechanism of TB formation and the effects of environmental stresses on coral survival and regeneration.

Prediction of the interaction site on the surface of an isolated protein structure by analysis of side chain energy scores.

We show that residues at the interfaces of protein-protein complexes have higher side-chain energy than other surface residues. Eight different sets of protein complexes were analyzed. For each protein pair, the complex structure was used to identify the interface residues in the unbound monomer structures. Side-chain energy was calculated for each surface residue in the unbound monomer using our previously developed scoring function.1 The mean energy was calculated for the interface residues and the other surface residues. In 15 of the 16 monomers, the mean energy of the interface residues was higher than that of other surface residues. By decomposing the scoring function, we found that the energy term of the buried surface area of non-hydrogen-bonded hydrophilic atoms is the most important factor contributing to the high energy of the interface regions. In spite of lacking hydrophilic residues, the interface regions were found to be rich in buried non-hydrogen-bonded hydrophilic atoms. Although the calculation results could be affected by the inaccuracy of the scoring function, patch analysis of side-chain energy on the surface of an isolated protein may be helpful in identifying the possible protein-protein interface. A patch was defined as 20 residues surrounding the central residue on the protein surface, and patch energy was calculated as the mean value of the side-chain energy of all residues in the patch. In 12 of the studied monomers, the patch with the highest energy overlaps with the observed interface. The results are more remarkable when only three residues with the highest energy in a patch are averaged to derive the patch energy. All three highest-energy residues of the top energy patch belong to interfacial residues in four of the eight small protomers. We also found that the residue with the highest energy score on the surface of a small protomer is very possibly the key interaction residue.

Evaluation of cytotoxicity, genotoxicity and embryotoxicity of insecticide propoxur using flounder gill (FG) cells and zebrafish embryos.

Cytotoxicity, genotoxicity and embryotoxicity of carbamate insecticide propoxur were evaluated using flounder gill (FG) cells and zebrafish embryos. The cytotoxicity of propoxur in FG cells was analyzed by MTT, neutral red uptake (NRU), lactate dehydrogenase (LDH) release and Hoechst 33342 and propidium iodide double staining, and acute cytotoxic effects were observed in a concentration-dependent manner. The 24h-IC50 values of 89.96 ± 1.04, 103.4 ± 1.14 and 86.59 ± 1.13 µg/ml propoxur were obtained by MTT, NRU and LDH assays, respectively. The lethal effects were induced in FG cells mainly through necrosis but not apoptosis as evidenced by double fluorescence staining. Comet assay showed weak genotoxic effects and statistically significant DNA damages were recorded in the cells exposed to highest tested concentration of 75 µg/ml propoxur (p<0.05). Propoxur exerted obvious acute toxic effects on the survival, spontaneous movement, hatching and heart rate, and development (yolk and pericardial sac edema) of zebrafish embryos in both time- and concentration-dependent manner only at ⩾ 100 µg/ml. The corresponding 24h-, 48 h- and 96 h-LC50 values of propoxur in zebrafish embryos were 166.4 ± 1.06, 146.3 ± 1.07 and 134.8 ± 1.06 µg/ml, respectively. The above data obtained suggest a low acute toxicity of propoxur to the in vitro cultured FG cells and zebrafish embryos.

Protein phosphatase 1 inhibits p53 signaling by dephosphorylating and stabilizing Mdmx.

The activation and stabilization of the p53 protein play a major role in the DNA damage response. Protein levels of p53 are tightly controlled by transcriptional regulation and a number of positive and negative posttranslational modifiers, including kinases, phosphatases, E3 ubiquitin ligases, deubiquitinases, acetylases and deacetylases. One of the primary p53 regulators is Mdmx. Despite its RING domain and structural similarity with Mdm2, Mdmx does not have an intrinsic ligase activity, but inhibits the transcriptional activity of p53. Previous studies reported that Mdmx is phosphorylated and destabilized in response to DNA damage stress. Three phosphorylation sites identified are Ser342, Ser367, and Ser403. In the present study, we identify protein phosphatase 1 (PP1) as a negative regulator in the p53 signaling pathway. PP1 directly interacts with Mdmx and specifically dephosphorylates Mdmx at Ser367. The dephosphorylation of Mdmx increases its stability and thereby inhibits p53 activity. Our results suggest that PP1 is a crucial component in the ATM-Chk2-p53 signaling pathway.

A novel function prediction approach using protein overlap networks.

BACKGROUND: Construction of a reliable network remains the bottleneck for network-based protein function prediction. We built an artificial network model called protein overlap network (PON) for the entire genome of yeast, fly, worm, and human, respectively. Each node of the network represents a protein, and two proteins are connected if they share a domain according to InterPro database. RESULTS: The function of a protein can be predicted by counting the occurrence frequency of GO (gene ontology) terms associated with domains of direct neighbors. The average success rate and coverage were 34.3% and 43.9%, respectively, for the test genomes, and were increased to 37.9% and 51.3% when a composite PON of the four species was used for the prediction. As a comparison, the success rate was 7.0% in the random control procedure. We also made predictions with GO term annotations of the second layer nodes using the composite network and obtained an impressive success rate (>30%) and coverage (>30%), even for small genomes. Further improvement was achieved by statistical analysis of manually annotated GO terms for each neighboring protein. CONCLUSIONS: The PONs are composed of dense modules accompanied by a few long distance connections. Based on the PONs, we developed multiple approaches effective for protein function prediction.

Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression.

p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renilla luciferase) into marine flatfish flounder gill (FG) cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C) and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation), but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl) phthalate (DEHP), a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner.

Evaluation of cytotoxicity, genotoxicity and teratogenicity of marine sediments from Qingdao coastal areas using in vitro fish cell assay, comet assay and zebrafish embryo test.

Marine sediments are often a final sink for numerous anthropogenic contaminants and may impose serious effects on benthic organisms and ecosystem. An in vitro cell assay using a cell line derived from flounder gill (FG) cells, an in vitro comet assay in FG cells, and an in vitro zebrafish embryo assay were used to evaluate the in vitro cytotoxicity (measured by MTT reduction), genotoxicity and teratogenicity of crude sediment extracts of Li Cang (LC), Zhan Qiao (ZQ) and Olympic Sailing Center (OSC) from Qingdao coastal area. Sediments from the three sites displayed different cytotoxicity, genotoxicity and teratogenicity potencies; however, all three assays yielded similar LOECs (lowest observed effect concentration) for each site, suggesting that the assays were equally sensitive to and suitable for initial screening of the LOECs of marine sediments. The cytotoxicity, genotoxicity and teratogenicity for these three sampling sites were in the same order of LC>ZQ>OSC, indicating different degrees of contamination. Interestingly, trials with the three sediment extracts at the doses inducing a similar cytotoxicity as evaluated with MTT reduction did not produce similar genotoxicity and teratogenicity, with the genotoxic and teratogenic activities of LC and ZQ extracts being markedly higher than those of OSC sediments. These findings indicate that cytotoxicity does not form a fully equivalent toxicity index with that of genotoxicity and teratogenicity. Therefore, in order to assess the true toxic potential of marine sediments, all three assays should be performed. Analysis of 16 EPA (US Environmental Protection Agency) priority PAHs in these three sediment samples showed a clear correlation between PAH concentrations and sediment toxicities, with a higher PAH content corresponding to higher toxicity although PAHs are surely not the only cause.

In vitro and in vivo acute toxicity of fenpyroximate to flounder Paralichthys olivaceus and its gill cell line FG.

Fenpyroximate, an acaricide, is widely used in the prevention of acarids (mites) in plants. In this study, the in vitro and in vivo acute toxicity of fenpyroximate was examined using the marine flounder Paralichthys olivaceus and its gill cell line (FG). The 48h-IC(50) (95% confidence limits) values of fenpyroximate in the FG cells were 890 (790-990)nM, 950 (881-1019)nM and 1250 (1159-1341)nM, and 96h-IC(50) (95% confidence limits) were 480 (388-572)nM, 490 (454-526)nM and 510 (469-551)nM, for methyl thiazolyl tetrazolium (MTT) assay, neutral red (NR) uptake and cell protein assay, respectively. The 48h- and 96h-LC(50) (95% confidence limits) values of fenpyroximate in living flounders were 28.84 (14.28-58.26)nM and 11.74 (6.06-22.8)nM, respectively. This indicated that fenpyroximate was highly toxic to both flounders and FG cells. Moreover, comparisons of the ratios of average 48h-IC(50) to 48h-LC(50) and average 96h-IC(50) to 96h-LC(50) showed that the length of exposure time did not significantly affect the correlation between the FG cells and living flounders in the acute toxicity estimation of fenpyroximate provided the selected exposure time is the same. Thus, we suggest that FG cells could be a good bioassay system in rapid estimation of the corresponding LC(50) values of pollutants to living fish, instead of whole living fish. Histopathological examinations showed that liver and gill were the major target organs of fenpyroximate, especially the damage of gill tissues may account much for the high lethality of exposed flounders. Consistent with the histopathological observations, analysis of the activities of two key detoxification metabolism-related enzymes, glutathione S-transferase (GST) and cytochrome P4501A1 (CYP1A1)-dependent ethoxyresorufin-O-deethylase (EROD), in the liver and gill tissues of exposed flounders indicated that liver has much higher detoxification capacity than gills, and this contributes to the higher tolerance of liver to the toxicity of fenpyroximate in the exposed flounders. Fenpyroximate can initially induce a quick and significant increase of the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in all the exposed FG cells, and liver and gill tissues of exposed flounders. Upon continuation of the exposure the enzyme activities were inhibited, implying the occurrence of oxidative stress in the exposed fish cells and the possible interruption of the mitochondrial respiratory chain which involves redox reactions by fenpyroximate.

Phosphorylation and degradation of MdmX is inhibited by Wip1 phosphatase in the DNA damage response.

MdmX and Mdm2 regulate p53 tumor suppressor functions by controlling p53 transcriptional activity and/or stability in cells exposed to DNA damage. Accumulating evidence indicates that ATM-mediated phosphorylation and degradation of Mdm2 and MdmX may be the initial driving force that induces p53 activity during the early phase of the DNA damage response. We have recently determined that a novel protein phosphatase, Wip1 (or PPM1D), contributes to p53 regulation by dephosphorylating Mdm2 to close the p53 activation loop initiated by the ATM/ATR kinases. In the present study, we determine that Wip1 directly dephosphorylates MdmX at the ATM-targeted Ser403 and indirectly suppresses phosphorylation of MdmX at Ser342 and Ser367. Wip1 inhibits the DNA damage-induced ubiquitination and degradation of MdmX, leading to the stabilization of MdmX and reduction of p53 activities. Our data suggest that Wip1 is an important component in the ATM-p53-MdmX regulatory loop.