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Molecules ◽  
2022 ◽  
Vol 27 (2) ◽  
pp. 344
Author(s):  
Adriana Trapani ◽  
María Ángeles Esteban ◽  
Francesca Curci ◽  
Daniela Erminia Manno ◽  
Antonio Serra ◽  
...  

The supply of nutrients, such as antioxidant agents, to fish cells still represents a challenge in aquaculture. In this context, we investigated solid lipid nanoparticles (SLN) composed of a combination of Gelucire® 50/13 and Precirol® ATO5 to administer a grape seed extract (GSE) mixture containing several antioxidant compounds. The combination of the two lipids for the SLN formation resulted in colloids exhibiting mean particle sizes in the range 139–283 nm and zeta potential values in the range +25.6–43.4 mV. Raman spectra and X-ray diffraction evidenced structural differences between the free GSE and GSE-loaded SLN, leading to the conclusion that GSE alters the structure of the lipid nanocarriers. From a biological viewpoint, cell lines from gilthead seabream and European sea bass were exposed to different concentrations of GSE-SLN for 24 h. In general, at appropriate concentrations, GSE-SLN increased the viability of the fish cells. Furthermore, regarding the gene expression in those cells, the expression of antioxidant genes was upregulated, whereas the expression of hsp70 and other genes related to the cytoskeleton was downregulated. Hence, an SLN formulation containing Gelucire® 50/13/Precirol® ATO5 and GSE may represent a compelling platform for improving the viability and antioxidant properties of fish cells.


2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Fei Ke ◽  
Xue-Dong Yu ◽  
Zi-Hao Wang ◽  
Jian-Fang Gui ◽  
Qi-Ya Zhang

Abstract Background Ranaviruses (family Iridoviridae) are promiscuous pathogens that can infect across species barriers in poikilotherms and can replicate in amphibian and fish cells and even in cultured mammalian cells. However, as nucleocytoplasmic large DNA viruses (NCLDVs), their replication and transcription mechanisms remain largely unknown. Here, we screened and uncovered the replication and transcription machinery of two ranaviruses, Andrias davidianus ranavirus (ADRV) and Rana grylio virus (RGV), by a combination of methods, including the isolation of proteins on nascent DNA, recombinant virus-based affinity, and NanoLuc complementation assay. Results The ranavirus replication and transcription machinery was deeply dissected and identified as a complicated apparatus containing at least 30 viral and 6 host proteins. The viral proteins ADRV-47L/RGV-63R (DNA polymerase, vDPOL), ADRV-23L/RGV-91R (proliferating cell nuclear antigen, vPCNA), ADRV-85L/RGV-27R (single-stranded DNA binding protein, vSSB), ADRV-88L/RGV-24R (vhelicase/primase), etc., constitute the core replisome. Specifically, the core of the transcription complex, the viral RNA polymerase, contain the host RNAPII subunits Rpb3, Rpb6, and Rpb11, which was a first report in NCLDVs. Furthermore, correlations and interactions among these factors in the machinery were described. Significantly, the replisome core protein vDPOL (ADRV-47L) can interact with numerous viral and host proteins and could act as a linker and regulation center in viral DNA replication and transcription. Thus, these results depicted an architecture for ranavirus replication and transcription. Conclusions Up to 36 components from ranavirus and their host were found to form viral replisomes and transcription complexes using a series of precise methods, which further constructed an architecture for ranavirus replication and transcription in which vDPOL was a key central factor and various components correlated and cooperated. Therefore, it provides a cornerstone for further understanding the mechanisms of the replication and transcription of ranaviruses which can ensure the efficient production of progeny virus and adaptation to cross-species infection.


Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1616
Author(s):  
Tuchakorn Lertwanakarn ◽  
Pirada Trongwongsa ◽  
Sangchai Yingsakmongkol ◽  
Matepiya Khemthong ◽  
Puntanat Tattiyapong ◽  
...  

The outbreak of the novel Tilapia tilapinevirus or Tilapia lake virus (TiLV) is having a severe economic impact on global tilapia aquaculture. Effective treatments and vaccines for TiLV are lacking. In this study, we demonstrated the antiviral activity of ribavirin against TiLV in E-11 cells. Our findings revealed that at concentrations above 100 μg/mL, ribavirin efficiently attenuates the cytopathic effect of the TiLV infection in fish cells. When administered in a dose-dependent manner, ribavirin significantly improved cell survival compared to the untreated control cells. Further investigation revealed that the cells exposed to ribavirin and TiLV had a lower viral load (p < 0.05) than the untreated cells. However, at concentrations above 1000 μg/mL, ribavirin led to cell toxicity. Taken together, our results demonstrate the efficacy of this antiviral drug against TiLV and could be a useful tool for future research on the pathogenesis and replication mechanism of TiLV as well as other piscine viruses.


2021 ◽  
Author(s):  
Beatriz Valenzuela ◽  
Almendra Benavides ◽  
Daniela Gutierrez ◽  
Lotsé Blamey ◽  
María Teresa Monsalves ◽  
...  

2021 ◽  
pp. 026119292110525
Author(s):  
Anaguiven Avalos-Soriano ◽  
Alejandra García-Gasca ◽  
Beatriz Yáñez-Rivera

Two cell lines derived from the brain and heart of a Pacific white snook specimen ( Centropomus viridis) were developed and evaluated in terms of their responsiveness to glyphosate-induced cytotoxicity. The cells were grown in Leibovitz-15 (L-15) medium supplemented with 10% fetal bovine serum (FBS) and were passaged 36 times. Growth was tested at different concentrations of FBS (5, 10 and 20%) at 27°C. The cell lines were cryopreserved at different passages and were successfully thawed, with a survival rate greater than 80% without detectable contamination. At passage 36, the cells were used to assess the deleterious effects of glyphosate, and cell proliferation was measured by direct counting and with the MTT assay. Similar LC50 values were obtained with both methods. Although the principles behind these two assessment methods differ, our results show that both are suitable for evaluating glyphosate toxicity. In addition, heart- and brain-derived cells showed similar sensitivity, suggesting that the same mode of action might be responsible for the toxicity of glyphosate at the cellular level. The newly developed Pacific white snook brain and heart cell lines could be useful to investigate cellular and molecular mechanisms of toxicity, satisfying the need to reduce the use of animals in experiments. Glyphosate-related toxicological data obtained in the present study will allow us to continue investigating the effects of this herbicide directly on brain and heart fish cells since similar studies have only been carried out on either live organisms or on human cell lines such as neuroblastoma, which are immortalised by oncogenes or similar.


Toxics ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 286
Author(s):  
Bianka Grunow ◽  
George Philipp Franz ◽  
Katrin Tönißen

Rising temperatures can affect fish survival, especially from shallower waters, as temperatures increase faster and more intensively in these areas; thus, species-specific temperature tolerance can be exceeded. Additionally, the amounts of anthropogenic pollutants are higher in coastal waters. Although increasing metabolic activity at higher temperatures could lead to stronger effects of toxins, there are hardly any studies on this topic. Subsequently, the aim was to investigate the response of fish cells upon exposure to industrial solvents (ethanol, isopropanol, dimethyl sulfoxide (DMSO)) in relation to a temperature increase (20 °C and 25 °C). Concerning the 3Rs (the replacement, reduction and refinement of animal experiments), in vitro tests were used for two threatened, vulnerable fish species: maraena whitefish (Coregonus maraena) and Atlantic sturgeon (Acipenser oxyrinchus). Both cell lines exhibited higher proliferation at 25 °C. However, ecotoxicological results indicated significant differences regarding the cell line, toxin, temperature and exposure time. The evolutionarily older fish lineage, Atlantic sturgeon, demonstrated lower mortality rates in the presence of isopropanol and recovered better during long-term ethanol exposure than the maraena whitefish. Atlantic sturgeon cells have higher adaptation potential for these alcohols. In summary, fish species respond very specifically to toxins and changes in temperature, and new ecotoxicological questions arise with increasing water temperatures.


2021 ◽  
Author(s):  
Marlana Malerich ◽  
Christopher Bryant

Abstract In recent decades, marine resources have faced extreme environmental pressures due to growing global fish consumption. Both commercial fishing and aquaculture harm the environment, threaten public health, and entail morally dubious practices. While consumers have increasingly become aware of the implications of the global fishing industry, most still want to eat seafood. Recent advancements in food technology have resulted in the successful production of cell-cultivated fish. Grown from real fish cells, cell-cultivated seafood avoids many of the issues associated with conventional fish production. Although cell-cultivated seafood will soon be available to consumers, there is not yet consensus on a ‘common or usual name’, a requirement of the US Food and Drug Administration for novel foods. We present a public discourse analysis, and the results of two online US-based surveys (n=2,452 and n=1,839) analyzing consumer acceptance and understanding of key terms used to describe cultured fish. Adult participants were tested for knowledge and acceptability of multiple descriptive terms: Bio-crafted, Bio-Cultivated, Cell-based, Cultivated, Cultured, Molecular, and the coined term ‘Novari’. The Control was a description of the product coupled with realistic packaging a consumer may expect to find once the product is available for purchase. The discourse analysis indicated that there is no current consensus on terminology used to describe cell-cultivated meat, and that some of the most commonly-used terms currently tend to be used in a negative context. Our Phase I survey revealed that names such as ‘cell-based’ and ‘bio-crafted’ were more likely to be understood, but relatively unappealing, while names such as ‘cultivated’ and ‘Novari’ were more appealing, but less likely to be understood. Our Phase II survey further revealed that the term ‘cell-cultivated’ combined promising elements of these terms, and was subsequently more appealing than ‘cell-based’ and better-understood than both ‘cultivated’ and ‘cell-based’. That said, none of the names tested outperformed the control group in consumer ability to identify the product accurately


Author(s):  
Hayato Nyunoya ◽  
Tatsuki Noda ◽  
You Kawamoto ◽  
Yasuhiro Hayashi ◽  
Yohei Ishibashi ◽  
...  

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jens Hamar ◽  
Dietmar Kültz

AbstractCRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.


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