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.

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.

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.

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%.

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.

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.

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.

ATF4 (activating transcription factor 4) from grass carp (Ctenopharyngodon idella) modulates the transcription initiation of GRP78 and GRP94 in CIK cells.

GRP78 and GRP94, belong to GRP (glucose-regulated protein) family of endoplasmatic reticulum (ER) chaperone superfamily, are essential for cell survival under ER stress. ATF4 is a protective protein which regulates the adaptation of cells to ER stress by modulating the transcription of UPR (Unfolded Protein Response) target genes, including GRP78 and GRP94. To understand the molecular mechanism of ATF4 modulates the transcription initiation of CiGRP78 and CiGRP94, we cloned ATF4 ORF cDNA sequences (CiATF4) by homologous cloning techniques. The expression trend of CiATF4 was similar to CiGRP78 and CiGRP94 did under 37 °C thermal stress, namely, the expression of CiATF4 was up-regulated twice at 2 h post-thermal stress and at 18 h post recovery from thermal stress. In this paper, CiATF4 was expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with the Ni-NTA His-Bind Resin. On the basis of the cloned CiGRP78 and CiGRP94 cDNA in our laboratory previously, we cloned their promoter sequences by genomic walking approach. In vitro, gel mobility shift assays revealed that CiATF4 could bind to CiGRP78 and CiGRP94 promoter with high affinity. Subsequently, the recombinant plasmid of pGL3-CiGRPs and pcDNA3.1-CiATF4 were constructed and transiently co-transfected into Ctenopharyngodon idella kidney (CIK) cells. The impact of CiATF4 on CiGRP promoter sequences were measured by luciferase assays. These results demonstrated that CiATF4 could activate the transcription of CiGRP78 and CiGRP94. What's more, for better understanding the molecular mechanism of CiATF4 modulate the transcription initiation of CiGRP, three mutant fragments of CiGRP78 promoter recombinant plasmids (called CARE-mut/LUC, CRE1-mut/LUC and CRE2-mut/LUC) were constructed and transiently co-transfected with CiATF4 into CIK cells. The results indicated that CRE or CARE elements were the regulatory element for transcription initiation of CiGRP78. Between them, CRE element would play more important role in it.

Enhanced electrochemical performance of MWNT@MnO2 composites in polymerized ionic liquids.

In this article, we report a newly devised composite electrode for electrochemical capacitors, by depositing manganese dioxide (MnO2) on the surface of carbon nanotubes via a spontaneous redox reaction. X-ray diffraction and transmission electron microscopic studies demonstrated a homogeneous coating of layered MnO2 on the surface of the carbon nanotubes. The MWNT@MnO2 composite electrode displayed enhanced capacity performance and rate capability as compared to the MnO2 electrode. Introduction of a thin layer of polymerized ionic liquid on the surface of the composite electrode enhanced the utilization rate of MnO2 and improved the conductivity of the resultant electrode. Electrochemical tests confirmed the augmented capacity performance and rate capabilities of the polyion-coated composite electrode.

The Zα domain of fish PKZ converts DNA hairpin with d(GC)(n) inserts to Z-conformation.

PKZ, protein kinase containing Z-DNA domains, is a novel member of the vertebrate eIF2α kinase family. Containing a catalytic domain in C-terminus and two Z-DNA binding domains (Zα1 and Zα2) in N-terminus, PKZ can be activated through the binding of Zα to Z-DNA. However, the regulatory function of PKZ Zα remains to be established. Here, to understand the impact of PKZ Zα on DNA conformational transition, wild-type Zα1Zα2 and 11 mutant proteins were expressed and purified. At the same time, several different lengths of DNA hairpins-d(GC)nT4(GC)n (n = 2-6) and an RNA hairpin-r(GC)6T4(GC)6 were synthesized. The effects of Zα1Zα2 and mutant proteins on the conformation of these synthetic DNA or RNA hairpins were investigated by using circular dichroism spectrum and gel mobility shift assays. The results showed that DNA hairpins retained a conventional B-DNA conformation in the absence of Zα1Zα2, while some of the DNA hairpins (n≥3) were converted to Z-conformation under Zα1Zα2 induction. The tendency was proportionally associated with the increasing amount of GC repeat. In comparison with Zα1Zα2, Zα1Zα1 rather than Zα2Zα2 displayed a higher ability in converting d(GC)6T4(GC)6 from B- to Z-DNA. These results demonstrated that Zα1 sub-domain played a more essential role in the process of B-Z conformational transition than Zα2 sub-domain did. Mutant proteins (K34A, N38A, R39A, Y42A, P57A, P58A, and W60A) could not convert d(GC)6T4(GC)6 into Z-DNA, whereas S35A or K56A retained some partial activities. Interestingly, Zα1Zα2 was also able to induce r(GC)6T4(GC)6 RNA from A-conformation to Z-conformation under appropriate conditions.

Overexpression of Hsp90 from grass carp (Ctenopharyngodon idella) increases thermal protection against heat stress.

With homologous DNA probes, we had screened a grass carp heat shock protein 90 gene (CiHsp90). The full sequence of CiHsp90 cDNA was 2793 bp, which could code a 798 amino acids peptide. The phylogenetic analysis demonstrated that CiHsp90 shared the high homology with Zebrafish Grp94. Quantitative RT-PCR analysis showed that CiHsp90 was ubiquitously expressed at lower levels in all detected tissues and up-regulated after heat shock at 34 °C or cold stress at 4 °C. To understand the function of CiHsp90 involving in thermal protection, an expression vector containing coding region cDNA was expressed in E. coli BL21 (DE3) plysS. Upon transfer from 37 °C to 42 °C, these cells that accumulated CiHsp90 peptides displayed greater thermoresistance than the control cells. While incubated at 4°C for different periods, it could also improve the cell viability. After transient transfected recombinant plasmid pcDNA3.1/CiHsp90 into mouse myeloma cell line SP2/0, we found that CiHsp90 could contribute to protecting cells against both thermal and cold extremes. On the contrary, the mutant construct ΔN-CiHsp90 (256-798aa) could abolish the protection activity both in prokaryotic cells and eukaryotic cells. Additionally, both CiHsp90 and ΔN-CiHsp90 peptides could reduce the level of citrate synthase aggregation at the high temperature.

Cloning and functional analysis of PKZ (PKR-like) from grass carp (Ctenopharyngodon idellus).

The new teleost fish PKZ (PKR-like) full-length cDNA (GU299765) had been cloned and identified from grass carp (Ctenopharyngodon idellus). The cDNA of grass carp PKZ (CiPKZ) has 2185 bp in length with a largest open reading frame (ORF) encoding 513aa. CiPKZ possesses a conserved C-terminal catalytic domain of eIF2α kinase family. Within its N-terminal there are two binding domain (Zα) named Zα1 (1-67aa) and Zα2 (81-152aa). BLAST homologous search reveals that CiPKZ has a high-level homology with other fish PKZs and PKRs. Like other fish PKZs and PKRs, CiPKZ is a ubiquitous tissue expression gene that had a very low level of constitutive expression but up-regulated in response to Poly I:C or hot stress (34 °C). For the purpose of searching for the potential function of CiPKZ, we obtained CiPKZ polypeptide via Escherichia coli Rosetta prokaryotic expression and purified with Ni-NTA His-Bind Resin affinity chromatography. CiPKZ polypeptide was used for the test of phosphorylating eIF2αin vitro. The results demonstrated that CiPKZ could be activated by Z-DNA but not by Poly I:C, and with subsequent could phosphorylate eIF2α. Meanwhile, four pcDNA3.1/PKZ recombinant plasmids, including pcDNA3.1/PKZ-wet, pcDNA3.1/PKZ-wet-K198R, pcDNA3.1/PKZ-wet-C, pcDNA3.1/PKZ-wet-C-K198R had been constructed, respectively. Mouse Myeloma cells (Sp2/0) and Human Umbilical Vein Endothelial Cells (HUVEC) were transiently cotransfected with pcDNA3.1/PKZ recombinant plasmid and PGL-3-promoter plasmid. The results revealed that CiPKZ could greatly decrease luciferase level in these cells. Zα and the K198 amino acid residue may play a key role in its function.

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.

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.

The Zalpha domain of PKZ from Carassius auratus can bind to d(GC)(n) in negative supercoils.

PKZ was the most recently discovered member of eIF2alpha kinase family in fish. CaPKZ, the first identified fish PKZ, possessed a conserved eIF2alpha kinase catalytic domain in C-terminal and two Z-DNA binding domains (Zalpha) in N-terminal. The Zalpha of CaPKZ closely resembled that of other Z-DNA binding proteins: ADAR1, DLM-1, and E3L. In order to understand more about the function of CaPKZ, we expressed and purified three constructed peptides of CaPKZ (P(Zalpha)): P(Zalpha1Zalpha2), P(Zalpha1Zalpha1) and P(Zalpha2)(Zalpha2). Moreover, most of the plasmids containing d(GC)(n) inserts were maintained in the Z-conformation, as confirmed by using inhibition of methylation experiments and anti-Z-DNA antibody. Gel mobility shift assays were then used to examine the affinity of these P(Zalpha) to the recombinant plasmids. Meanwhile, a competition experiment using P(Zalpha1Zalpha2) and anti-Z-DNA antibody was performed. The results revealed that P(Zalpha1Zalpha2) and P(Zalpha1Zalpha1) were able to bind to the recombinant plasmids with high affinity, whereas P(Zalpha2)(Zalpha2) could not bind to it. In addition, dimerization of P(Zalpha1Zalpha2) indicated the function unit of Zalpha of CaPKZ would be a dimer.

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.

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.