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.

Cloning and functional analysis of SNX8 from grass carp (Ctenopharyngodon idellus).

Sorting nexin 8 (SNX8), a member of sorting nexin protein family, plays important roles in endocytosis, endosomal sorting, and innate immune response. To date, a few homologs of SNX8 have been found in fish except in mammals. In this study, a teleost SNX8 cDNA was identified from grass carp (Ctenopharyngodon idellus). CiSNX8 was up-regulated significantly after infection with poly I:C or GCRV. We found that SNX8 was mainly distributed in the endoplasmic reticulum (ER) in CIK cells. Further analysis indicated that CiSNX8 might negatively regulate RLR signaling pathway that is quite distinct from mammalian SNX8. In addition, CiSNX8 could interact with MAVS, STING, TBK1, IRF3 and IRF7. Either wild type CiSNX8 or mutants of N-terminal PX domain (aa 1-245) and C-terminal BAR domain (aa 256-519) could associate with STING. These results suggested that fish SNX8 participated in innate immune response through different molecular mechanisms.

Laser-direct-writing of molecule-like Agmx+ nanoclusters in transparent tellurite glass for 3D volumetric optical storage.

In situ constructing program-designed nanostructures via laser-direct-writing (LDW) has proved to be a reliable strategy for optical storage (OS). Herein, a kind of low-melting Ag+-doped TeO2-ZnO-Na2O (TZN) tellurite glass has been demonstrated as an ideal LDW OS medium. Microstructural and spectroscopic studies reveal the generation of molecule-like Agmx+ nanoclusters featured with a broad emission band in the orange-red region upon laser irradiation. Probing the laser-glass interaction yields evidences of the spatial distribution of Ag species responsive to laser-induced thermoelastic pressure wave oscillation, as well as the heat-driven migration/aggregation of Ag species along the radial direction of the laser spot. Raman analyses disclose the loose network of TZN-glass convenient for Ag+ mobility and the increased network connectivity when Agmx+ nanoclusters are precipitated out. Combined with the XPS result of Ag+ → Ag0 reduction, the possible formation mechanism of Ag nanoclusters stabilized in glass has been proposed. In a proof-of-concept experiment, 3D volumetric OS in the TZN glass has been demonstrated, showing optical data encoding/decoding in the form of characters and image patterns.

Ctenopharyngodon idella Tollip regulates MyD88-induced NF-κB activation.

Toll-interacting protein (Tollip) and MyD88 are key components of the TLR/IL-1R signaling pathway in mammals. MyD88 is known as a universal adaptor protein involving in TLR/IL-1R-induced NF-κB activation. Tollip is a crucial negative regulator of TLR-mediated innate immune responses. Previous studies have demonstrated that teleost Tollip served as a negative regulator of MyD88-dependent TLR signaling pathway. However, the mechanism is still unclear. In particular, the effect of TBD, C2, and CUE domains of Tollip on MyD88-NF-κB signaling pathway remains to be elucidated. In this study, we found that the response of grass carp Tollip (CiTollip) to LPS stimulation was faster and stronger than that of poly I:C treatment, and CiTollip diminished the expression of tnf-α induced by LPS. Further assays indicated that except for the truncated mutant of △CUE2 (1-173 aa), wild type CiTollip and other truncated mutants (△N-(52-276 aa), △C2-(173-276 aa) and △CUE1-(1-231 aa)) could associate with MyD88 and negatively regulate MyD88-induced NF-κB activation. It suggested that the C-terminal (173-276 aa), in particular the connection section between C2 and CUE domains (173-231 aa), played a pivotal role in suppressing MyD88-induced activation of NF-κB.

Uniform Pt nanoparticles supported on urchin-like mesoporous TiO2 hollow spheres as stable electrocatalysts for the oxygen reduction reaction.

In order to promote the commercial application of proton exchange membrane fuel cells, it is of great importance to develop Pt-based electrocatalysts with high activity and stability for the oxygen reduction reaction (ORR). Here, urchin-like mesoporous TiO2 hollow spheres (UMTHS) with a high specific surface area (167.1 m2 g-1) and improved conductivity were designed and applied as supports to disperse Pt nanoparticles (NPs) for the first time. Uniform Pt NPs (∼3.2 nm) on the surface of nanothorns were obtained after heat treatment. The as-prepared product (Pt/UMTHS) exhibited a more positive half-wave potential (Eh) than that of the reference sample Pt@C without UMTHS (0.867 V vs. 0.829 V). The improved performance can be ascribed to the high specific surface area of UMTHS. The Pt/UMTHS also exhibited a much better ORR stability than the commercial Pt/C after long-term cycling at 0.6-1.0 V according to the comparison of Eh, mass activity and electrochemical surface area with Pt/C. The enhanced stability of Pt/UMTHS was mainly derived from the strong metal support interaction between Pt NPs and UMTHS, together with the spatial restriction and the anti-restriction provided by UMTHS.

PKZ, a Fish-Unique eIF2α Kinase Involved in Innate Immune Response.

PKZ is a novel and unique eIF2α protein kinase identified in fish. Although PKZ is most homologous to PKR, particularly in the C-terminal catalytic domain, it contains two N-terminal Z-DNA-binding domains (Zα1 and Zα2) instead of the dsRNA binding domains (dsRBDs) in PKR. As a novel member of eIF2α kinase family, the available data suggest that PKZ has some distinct mechanisms for recognition, binding, and B-Z DNA transition. Functionally, PKZ seems to be activated by the binding of Zα to Z-DNA and participates in innate immune responses. In this review, we summarize the recent progress on fish PKZ.

Grass carp (Ctenopharyngodon idellus) TRAF6 up-regulates IFN1 expression by activating IRF5.

In mammals, interferon regulatory factor 5 (IRF5) can be activated by tumor necrosis factor receptor-associated factor 6 (TRAF6). Upon activation, IRF5 translocates into the nucleus, where it binds to IFN promoter and up-regulates IFN expression. However, there are few reports on the molecular mechanism by which TRAF6 up-regulates IFN expression in fish. In this study, we explored how Grass carp (Ctenopharyngodon idellus) TRAF6 initiated innate immunity by activating IRF5. We found that CiTRAF6, CiIRF5 and CiIFN1 were all significantly up-regulated in LPS-stimulated CIK cells and the expression of CiTRAF6 was earlier than the expressions of CiIRF5 and CiIFN1. These findings suggested that CiIFN1 expression might be induced by CiTRAF6 in fish. CiIFN1 expression, CiIFN1 promoter activity and CO cells viability were all significantly up-regulated in the overexpression experiments, but they were significantly down-regulated in the gene silencing experiments. This indicated that CiTRAF6, along with CiIRF5, regulated CiIFN1 expression. The localization analysis found that both CiTRAF6 and CiIRF5 located in the cytoplasm. Following LPS stimulation, CiIRF5 was observed to translocate to the nucleus. GST-pull down and co-IP experiments revealed that CiTRAF6 interacted with CiIRF5. The colocalization analysis also showed that CiTRAF6 bound with CiIRF5 in the cytoplasm. Overexpression of CiTRAF6 increased the endogenous CiIRF5, promoted its ubiquitination and nuclear translocation. In conclusion, CiTRAF6 bound to CiIRF5 in the cytoplasm, and then activated CiIRF5, resulting in up-regulating the expression of CiIFN1.

An Fe-N co-doped tube-in-tube carbon nanostructure used as an efficient catalyst for the electrochemical oxygen reduction reaction.

An Fe-N co-doped tube-in-tube carbon nanostructure is synthesized for an efficient oxygen reduction reaction. Thanks to its hollow nature, the mesoporous structure is enriched while defects are not prominent, allowing excellent activity (E 1/2 = 0.851 V) and durability together with methanol tolerance in an electrochemistry test under alkaline conditions. Furthermore, when the material is used as the cathode catalyst of a Zn-air battery, the battery exhibits a peak power density of 181.5 mW cm-2.

Ctenopharyngodon Idella STAT3 alleviates autophagy by up-regulating BCL-2 expression.

In mammals, STAT3 (Signal transducer and activator of transcription 3) plays an absolutely vital role in response to cytokines and growth factors. In mammals, IL-6/JAK/STAT3 pathway is closely linked to immune response and promotes cell proliferation, survival and metastasis. Some recent studies have already demonstrated that STAT3 regulates autophagy. As a downstream target gene of STAT3, Bcl-2 (B-cell lymphoma 2) not only participates in regulating apoptosis, but also responds to autophagy. STAT3 regulates autophagy through Bcl-2. In general, the generation of autophagy is always accompanied by the change of apoptosis, and the occurrence of apoptosis is often accompanied by the decreased of cell viability. In grass carp (Ctenopharyngodon idella), LPS-induced autophagy is involved in the release of pro-inflammatory cytokines. However, only the relationship between autophagy and cytokines was illustrated, in which the signaling pathways were not discussed. In the present study, we found that the autophagy inducer, Tunicamycin (Tm), can induce C.Idella Kidney cells (CIK) autophagy. When the cells were incubated with the recombinant human IL-6 (rIL-6) for a short period of times, the mRNA expression level of C.Idella IL-6R and STAT3 were increased. At the same time, the number of GFP-LC3 puncta and the ratio of LC3-II/LC3-I were both decreased obviously in cells. It indicated that the rIL-6 can significantly alleviate autophagy induced by Tm. We speculated that CiSTAT3 may play a key role in the process. To confirm this hypothesis, we performed a rIL-6 activating CiSTAT3 assay. The result demonstrated that rIL-6 can induce CiSTAT3 to form homologous dimmer. The activated CiSTAT3 regulated the transcription activity of CiBcl-2, finally led to a decrease of autophagy. In addition, when cells were in the state of autophagy, apoptosis was increased and cell viability was decreased. When CiSTAT3 was activated, cell apoptosis weakened and cell viability was increased. The results suggest that CiSTAT3 plays an important role in maintaining the normal physiological process of cells.

The Fish-Specific Protein Kinase (PKZ) Initiates Innate Immune Responses via IRF3- and ISGF3-Like Mediated Pathways.

PKZ is a fish-specific protein kinase containing Zα domains. PKZ is known to induce apoptosis through phosphorylating eukaryotic initiation factor 2α kinase (eIF2α) in the same way as double-stranded RNA-dependent protein kinase (PKR), but its exact role in detecting pathogens remains to be fully elucidated. Herein, we have found that PKZ acts as a fish-specific DNA sensor by initiating IFN expression through IRF3- or ISGF3-like mediated pathways. The expression pattern of PKZ is similar to those of innate immunity mediators stimulated by poly (dA:dT) and poly (dG:dC). DNA-PKZ interaction can enhance PKZ phosphorylation and dimerization in vitro. These findings indicate that PKZ participates in cytoplasmic DNA-mediated signaling. Subcellular localization assays have also shown that PKZ is located in the cytoplasm, which suggests that PKZ acts as a cytoplasmic PRR. Meanwhile, co-IP assays have shown that PKZ can separately interact with IRF3, STING, ZDHHC1, eIF2α, IRF9, and STAT2. Further investigations have revealed that PKZ can activate IRF3 and STAT2; and that IRF3-dependent and ISGF3-like dependent mediators are critical for PKZ-induced IFN expression. These results demonstrate that PKZ acts as a special DNA pattern-recognition receptor, and that PKZ can trigger immune responses through IRF3-mediated or ISGF3-like mediated pathways in fish.

High-efficient and environmentally friendly enrichment of semiconducting single-walled carbon nanotubes by combining short-time electrochemical pre-oxidation and combustion.

High purity semiconducting single-walled carbon nanotubes (s-SWCNTs) have bright prospects in the field of microelectronics, but their enrichment processes are usually very complicated and cost time and energy, which represent a major impediment for their future applications. Here, we report on a new efficient covalent modification enrichment approach that tackles this problem. Our method is to first selectively functionalize the surface of arc-discharge metallic single-walled carbon nanotubes (m-SWCNTs) rapidly by electrochemical pre-oxidation at 7.0 V in 0.1 M KCl aqueous solution, and subsequently followed up by removing the m-SWCNTs with a short-time combustion process at 600 °C for 30 s to enrich high purity s-SWCNTs. Although the surface of the s-SWCNTs was functionalized and heat-treated, the intrinsic tubular structure and electronic characteristics were well maintained. Besides, our approach, without any complex equipment or toxic reagents, is energy and time saving and can be easily scaled up. Milligrams of high-quality s-SWCNTs with high purity of more than 95 wt% can be easily obtained in only several minutes. The retention rate of s-SWCNTs after combustion is as high as 61 wt%.

Identification of the SAMHD1 gene in grass carp and its roles in inducing apoptosis and inhibiting GCRV proliferation.

SAMHD1 is an innate immunity restriction factor that inhibits virus infection through IRF3-mediated antiviral and apoptotic responses. Fish SAMHD1 shares some similar properties with those in mammals. In this study, a SAMHD1 orthologue from grass carp (Ctenopharyngodon idellus) was cloned and characterized. The full-length cDNA of CiSAMHD1 is 2792 bp with an ORF of 1884 bp encoding a polypeptide of 627 amino acids. Multiple alignments showed that SAMHD1 is highly conserved among different species. Phylogenetic tree analysis revealed that CiSAMHD1 shared a high degree of homology with Sinocyclocheilus rhinocerous SAMHD1. Expression analysis indicated that CiSAMHD1 was widely expressed in all tissues tested including the brain, eyes, spleen, gill, intestine, liver, heart and kidney. It was significantly up-regulated in spleen, liver and intestines after treatment with poly I:C. Also, CiSAMHD1 can be induced following stimulation with recombinant IFN in CIK cells. The promoter sequence of CiSAMHD1 was identified to explore the mechanism underlying the transcriptional regulation of CiSAMHD1. The promoter sequence of CiSAMHD1 (1370 bp) consists of IRF1, IRF3, IRF9 and p65 binding elements. Gel mobility shift assay also showed that IRF1, IRF3, IRF9 and p65 prokaryotic proteins can separately interact with CiSAMHD1 promoter. Dual luciferase assay and q-PCR suggested that the promoter of CiSAMHD1 can be activated by the overexpression of CiIRF3 and CiIRF9, but cannot be triggered by CiIRF1 and Cip65. In contrast, knockdown of CiIRF3 or CiIRF9 inhibits the transcription of CiSAMHD1. Intriguingly, CCK assay suggested that CiSAMHD1 decreased cell viability. TUNEL apoptosis assay and Hoechst 33258 staining assay indicated that apoptosis is induced by the overexpression of CiSAMHD1. Crystal violet staining, detection of two GCRV genes (vp3 and vp5) and viral titration showed that CiSAMHD1 can suppress the proliferation of grass carp reovirus (GCRV) in CIK cells.

Identification and functional characterization of IRAK-4 in grass carp (Ctenopharyngodon idellus).

IL-1R-associated kinase 4 (IRAK4), a central TIR signaling mediator in innate immunity, can initiate a cascade of signaling events and lead to induction of inflammatory target gene expression eventually. In the present study, we cloned and characterized an IRAK4 orthologue from grass carp (Ctenopharyngodon idella). The full length cDNA of CiIRAK4 was 2057 bp with an ORF of 1422 bp encoding a polypeptide of 472 amino acids. Multiple alignments showed that IRAK4s were highly conserved among different species. Phylogenetic tree analysis revealed that CiIRAK4 shared high homologous with zebra fish IRAK4. Expression analysis indicated that CiIRAK4 was widely expressed in all tested tissues. It was significantly up-regulated after treatment with poly I:C, especially obvious in liver and spleen. Also, CiIRAK4 could be induced by poly I:C and LPS in CIK cells. Fluorescence microscopy assays showed that CiIRAK4 localized in the cytoplasm. RNAi-mediated knockdown and overexpression assays indicated that CiIRAK4 might have little effect on NF-kappa B p65 translocation from cytoplasm to nucleus, indicating that CiIRAK4 was dispensable for activation of NF-kappa B p65. In addition, IRAK4 promoted IRF5 nuclear translocation, which has nothing to do with the interaction between IRAK4 and IRF5. It suggested that fish IRAK4 kinase regulated IRF5 activity through indirect ways.

Enrichment of semiconducting single-walled carbon nanotubes by simple equipment and solar radiation.

High-purity semiconducting (s-) single-walled carbon nanotubes (SWCNTs) have great potential to replace silicon-based materials for microelectronic devices. However, the enrichment methods of s-SWCNTs usually required complex devices and non-renewable energy. In this study, instead of a traditional heating method, renewable solar was employed to dramatically increase the heating rate and improve the reaction to be simple and more controllable, thereby water was successfully used to selectively etch metallic (m-) SWCNTs. In this work, purified SWCNTs films were wetted by water and then exposed to focused solar radiation, causing the surface temperature of the SWCNT films to reach about 800 °C within 2 s. In this case, the m-SWCNTs could be selectively etched by water rapidly. Finally, s-SWCNTs with a purity of about 95 wt% were obtained in several minutes without any complex devices or non-renewable energy.

Iron encapsulated in single-walled carbon nanotubes for obtaining the evidence of improved coulombic efficiency and improving the lithium battery performance of ZnO anodes.

Cycling coulombic efficiency including the 1st cycle is a crucial factor for nano-carbon based anodes. How to improve their coulombic efficiency and further prove whether the additional reversible capacity produced from the SEI film in the 1st cycle is an obstacle for their possible commercial application in Li ion batteries (LIBs). For this aim, a novel composite of Fe-encapsulated single-walled carbon nanotubes (Fe@SWNTs) with special nano-structure was designed and used as an anode material for LIBs. The resulting Fe@SWNT anode can provide much larger coulombic efficiency of 53.1% in the 1st cycle than 35.6% for pure SWNTs, implying the value increment reached ∼50%. The Fe@SWNTs can exhibit an reversible capacity of 420 mA h g-1 after 300 cycles and excellent rate performance at room temperature, being obviously better than 275 mA h g-1 for a SWNT anode. The origination of this extra improved reversible capacity can be confirmed to be derived from the reversible reaction of SEI film activated by the Fe catalyst. Meanwhile, the Fe@SWNT anodes exhibited superior low-temperature (at 5 and -15 °C) electrochemical performance, which should be associated with an improved effect of the highly conducting Fe at low temperature, and with the activation of catalyst Fe on the reversible capacity. In addition, when Fe@SWNTs were developed as carriers for attaching ZnO, the ZnO/Fe@SWNTs can deliver much better LIB performance than anodes of pure ZnO and ZnO/SWNTs. Thus, catalyst modification supplied a promising route to obtain improved coulombic efficiency and reversible capacity for LIB nano-carbon based anodes.

Grass carp (Ctenopharyngodon idella) STAT3 regulates the eIF2α phosphorylation through interaction with PKR.

In mammals, STAT3 (Signal transducer and activator of transcription 3) plays an important role in growth, multiplication, differentiation and participates in inflammation, tumorigenesis, metabolic disorders and immune response. STAT3 is a protein that shuttles between the nucleus and cytoplasm. Compared to the STAT3 in cell nucleus, we did not know the function of STAT3 in cytoplasm for a long time. Some recent studies have shown that cytoplasmic STAT3 regulates autophagy through the interaction with the double-stranded RNA-activated protein kinase (PKR), which plays an important role in cellular antiviral response. Fish is a good target for developmental and comparative immunology. In the present study, we found that the expression of grass carp (Ctenopharyngodon idella) STAT3 (CiSTAT3) was ubiquitous and significantly up-regulated under the stimulation of poly I:C. To explore the potential function of fish cytoplasmic STAT3 in the antiviral signaling pathways, in this paper we analyzed the relationship between cytoplasmic CiSTAT3 and CiPKR. We demonstrated that the CiSTAT3 can combine with CiPKR in vivo and in vitro. The SH2 domain of CiSTAT3 and the C-terminus of CiPKR play an important role in this process. Moreover, the dimer of CiSTAT3 and CiPKR was formed under normal circumstances, however, it was dissociated under the induction of poly I:C. So, we guessed the binding of CiSTAT3 and CiPKR may regulate cell viability. It has also been shown that overexpression of CiSTAT3 in CIK cells can significantly reduce the level of p-eIF2α. On the contrary, the siRNA-mediated knockdown of CiSTAT3 and Stattic induction in CIK cells can up-regulate the p-eIF2α level. To further understand the relationship between CiSTAT3 and p-eIF2α level, we carried out the CiPKR-knockdown experiment. The result indicated that CiSTAT3 regulated the level of p-eIF2α through binding to CiPKR. In addition, overexpression of CiSTAT3 in CIK cells was able to improve the cell viability. These results above unraveled the molecular mechanism of fish cytoplasmic STAT3 regulating the eIF2α phosphorylation and cell viability. Therefore, the function of fish cytoplasmic STAT3 is similar to those of mammals.

Pt Nanoparticles Densely Coated on SnO2-Covered Multiwalled Carbon Nanotubes with Excellent Electrocatalytic Activity and Stability for Methanol Oxidation.

A new electrocatalyst exhibiting enhanced activity and stability is designed from SnO2-covered multiwalled carbon nanotubes coated with 85 wt % ratio Pt nanoparticles (NPs). This catalyst showed a mass activity 6.2 times as active as that of the commercial Pt/C for methanol oxidation, owing to the unique one-dimensional structure. Moreover, the durability and antipoisoning ability were also improved greatly. The enhanced intrinsic performance was ascribed to the densely connected networks of Pt NPs on the SnO2 NPs.

MnO2 Nanofilms on Nitrogen-Doped Hollow Graphene Spheres as a High-Performance Electrocatalyst for Oxygen Reduction Reaction.

Platinum is commonly chosen as an electrocatalyst used for oxygen reduction reaction (ORR). In this study, we report an active catalyst composed of MnO2 nanofilms grown directly on nitrogen-doped hollow graphene spheres, which exhibits high activity toward ORR with positive onset potential (0.94 V vs RHE), large current density (5.2 mA cm-2), and perfect stability. Significantly, when it was used as catalyst for air electrode, a zinc-air battery exhibited a high power density (82 mW cm-2) and specific capacities (744 mA h g-1) comparable to that with Pt/C (20 wt %) as air cathode. The enhanced activity is ascribed to the synergistic interaction between MnO2 and the doped hollow carbon nanomaterials. This easy and cheap method paves a way of synthesizing high-performance electrocatalysts for ORR.

Direct Enrichment of Metallic Single-Walled Carbon Nanotubes by Using NO2 as Oxidant to Selectively Etch Semiconducting Counterparts.

We report an efficient method for enriching high-purity metallic single-walled carbon nanotubes (m-SWCNTs) by using NO2 as oxidant to remove semiconducting components at 220 °C. After etching, m-SWCNTs with purity higher than 90% were obtained. The surviving m-SWCNTs retain an intact structure without any extra defects on their surface.

Identification and function analysis of the three dsRBMs in the N terminal dsRBD of grass carp (Ctenopharyngodon idella) PKR.

The protein kinase R (PKR) can inhibit protein translation and lead to apoptosis under the circumstances of virus invasion and multiple other stress conditions. PKR is a dsRNA binding protein with a dsRBD and a kinase domain (KD). dsRBD is mostly composed of two (in mammal PKR) or three (in some fish PKR) dsRNA binding motifs (dsRBMs). Multiple sequences alignment and Phylogenetic analysis indicate that the three dsRBMs of fish PKR share analogous structure but show to be divergence origination. In this study, we have identified and analyzed the three dsRBMs from grass carp (Ctenopharyngodon idellus) PKR (CiPKR), which was cloned previously in our laboratory. dsRBMs of CiPKR have two or three conserved regions involved in dsRNA binding. Among the three dsRBMs, dsRBM1 was peculiar to some fish PKRs, while dsRBM2 and dsRBM3 were closely related to the dsRBM1 and dsRBM2 of mammal PKRs respectively. Dimerization assay indicated that dsRBM1 and dsRBM2 formed not only homo-dimer but also homo-multimer; whereas dsRBM3 formed merely homo-dimer. Meanwhile, dsRBM1-2, dsRBM2-3 and dsRBM1-2-3 could homo-dimerize and homo-multimerize also. Poly I:C pull-down assay showed that the binding of dsRBM to Poly I:C needed two or three dsRBMs to cooperate in vitro, meaning one dsRBM from CiPKR could not bind to dsRNA efficiently. To further investigate the effect of dsRBM on the function of CiPKR, we constructed pcDNA3.1/CiPKR-wt and a series of CiPKR mutants recombined plasmids including pcDNA3.1/CiPKR-ΔdsRBM2-3, pcDNA3.1/CiPKR-ΔdsRBM1,3, pcDNA3.1/CiPKR-ΔdsRBM1-2, pcDNA3.1/CiPKR-ΔdsRBM3, pcDNA3.1/CiPKR-ΔdsRBM1. The recombined plasmids respectively were co-transfected with plasmid PGL3 promoter into CIK cells. In comparison with the control group, the luciferase translation inhibitions were 78.7%, 15%, 0, 0.5%, 61.8%, 67.3% respectively. The results indicated that the protein translation inhibition caused by CiPKR mutants with only one dsRBM were very weak, while those with two or three dsRBMs inhibited the protein translation powerfully. Cell viability were 34.2%, 98.2%, 112%, 108%, 50.3%, 47.5% respectively after transfected with pcDNA3.1/CiPKR-wt, pcDNA3.1/CiPKR-ΔdsRBM2-3, pcDNA3.1/CiPKR-ΔdsRBM1,3, pcDNA3.1/CiPKR-ΔdsRBM1-2, pcDNA3.1/CiPKR-ΔdsRBM3, pcDNA3.1/CiPKR-ΔdsRBM1 in order into CIK cells for 48 h. The results from cell counting also indicated that transfection of CiPKR-wt and the mutants CiPKR-ΔdsRBM3, CiPKR-ΔdsRBM1 could inhibit the protein translation and facilitated the decrease of CIK cells number. In conclusion, our observations suggested that two dsRBMs ranking in tandem at N terminal were essential for the function of CiPKR, and the presence of the extra dsRBM1 enhanced its function.