Grass carp (Ctenopharyngodon idella) NRF2 alleviates the oxidative stress and enhances cell viability through upregulating the expression of HO-1.

As a member of the Cap 'n' Collar (CNC) family, NRF2 contains a basic leucine zipper (bZip) and can regulate the downstream target gene heme oxygenase 1 (HO-1) in response to oxidative stress. In the present study, a grass carp (Ctenopharyngodon idella) NRF2 ORF was cloned and identified. The largest ORF (1782 bp) encodes a polypeptide of 593 amino acids. The deduced amino acid sequence of grass carp NRF2 (CiNRF2) contains a well-conserved DNA-binding domain (BRLZ domain). Phylogenetic tree analysis revealed that CiNRF2 has a closer evolutionary relationship with other fish counterparts. After CIK (C. idellus kidney) cells were persistently stimulated with tunicamycin (TM), CiNRF2 was significantly upregulated from 12 to 36 h. Then, the expression was dropped at 48 h post-infection. Additionally, when TM or TG (thapsigargin) stimulated CIK cells, overexpression of CiNRF2 in cells downregulated the expression of Bip mRNA, a marker protein of oxidative stress, suggesting that fish NRF2 can alleviate the oxidative stress level induced by TM or TG. To study the protective mechanism of fish NRF2, the DNA sequences of CiNRF2 and CiATF4 (grass carp ATF4) were separately sub-cloned into the expression vectors pEGFP and pCMV-Flag for co-immunoprecipitation and GST pull-down assays. These assays showed that CiNRF2 can combine with CiATF4 through its Neh1 domain. Meanwhile, we cloned grass carp HO-1 promoter sequence and constructed the recombinant plasmid of pGL3-HO-1. Soon afterwards, pGL3-HO-1 was co-transfected into grass carp ovary (CO) cells with pcDNA3.1-CiNRF2 or pcDNA3.1-CiATF4, respectively. The results showed that the luciferase activity of pGL3-HO-1 in the overexpressed CiNRF2 plus CiATF4 cells was significantly increased, along with the increase of cell viability (~ 133%). However, when HO-1 was knocked down in cells, CiNRF2 was unable to perform its function. These results demonstrated that CiNRF2 was effective in protecting grass carp against the oxidative stress induced by TM and increasing cell viability by upregulating HO-1 expression.

Grass carp (Ctenopharyngodon idella) Bcl-xl: transcriptional regulation and anti-apoptosis analysis.

Bcl-xl, Bax2, and NF-κB are well-known to be involved in anti-apoptosis response. Although Bcl-xl has been reported in fish, the NF-κB-mediated regulatory mechanism and anti-apoptotic function are still unclear. Here, we cloned and characterized the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) Bcl-xl (CiBcl-xl) and its promoter region sequence. The full-length cDNA of CiBcl-xl is 2836 bp with an ORF of 627 bp encoding a polypeptide of 208 amino acids. Phylogenetic tree analysis revealed that CiBcl-xl shared high homology with Dario rerio Bcl-xl (DrBcl-xl). After stimulation with Poly I:C, the expression of CiBcl-xl in CIK cells and various tested tissues of grass carp were significantly upregulated. To further understand the transcriptional control of fish Bcl-xl induced by NF-κB, CiC-rel and Cip65 were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. In vitro, gel mobility shift assays demonstrated the high affinity of CiC-rel and Cip65 with CiBcl-xl promoter. Dual-luciferase reporter assays showed that CiC-rel and Cip65 activated CiBcl-xl promoter. Also, knockdown of CiC-rel and Cip65 reduced the expression of Bcl-xl. Therefore, similar to those of mammals, fish C-rel and p65 can upregulate the transcription of Bcl-xl. In addition, we found that overexpression of CiBcl-xl in CIK cells increased the cell activity and inhibited cell apoptosis, while overexpression of Bax2 promoted cell apoptosis. Meanwhile, co-transfection of CiBcl-xl and CiBax2 into cells can ease up apoptotic rate. To further investigate the molecular basis of synergistic effect of Bcl-xl and Bax2, we showed that Bcl-xl and Bax2 interacted with each other. The results suggested that Bcl-xl executed its anti-apoptotic function by binding to and inhibiting the pro-apoptotic activity of Bax2.

Ctenopharyngodon idella TBK1 activates innate immune response via IRF7.

In mammals, IFN regulatory factor (IRF) 7 is a central regulator of IFN-α expression in response to variable pathogenic infections. There are several pathogenic sensors involved in monitoring pathogen intrusion in mammals. These sensors trigger IRF7-mediated responses through different pathways. TANK-binding kinase 1 (TBK1) is a critical mediator of IRF7 activation upon pathogen infection. In fish, there are many reports on TBK1, IRF3 and IRF7, especially on TBK1-IRF3 signaling pathway. However, it is not very clear how TBK1-IRF7 works in innate immune signaling pathway. In this study, we explored how TBK1 up-regulates IFN, ISG expression, and how TBK1 initiates innate immune response through IRF7 in fish under lipopolysaccharides (LPS) stimulation. After stimulation with LPS, grass carp IRF3 and IRF7 transcriptions were up-regulated, indicating they participate in TLR-mediated antiviral signaling pathway. It is interesting that the response time of grass carp IRF3 to LPS was earlier than that of IRF7. In addition, IRF7 rather than IRF3 acted as a stronger positive regulator of IFN and ISG transcription in Ctenopharyngodon idella kidney cells (CIKs). It is suggested the potential function differentiation between IRF3 and IRF7 upon LPS infection in fish. Dual luciferase assays also showed that overexpression of grass carp IRF7 and TBK1 up-regulated the transcription level of IFN and PKR. However, knockdown of IRF7 inhibits ISG expression, suggesting that grass carp TBK1 regulates the transcription via IRF7. Co-immunoprecipitation and GST pull-down assays proved the binding of grass carp IRF7 to TBK1. Furthermore, grass carp TBK1 can promote the nuclear translocation of IRF7. The results indicated that grass carp TBK1 can bind directly to and activate IRF7.

Activated grass carp STAT6 up-regulates the transcriptional level and expression of CCL20 and Bcl-xl.

In mammals, signal transducer and activator of transcription 6 (STAT6) is a broad-spectrum transcriptional regulator involved in cellular immune responses and apoptosis by regulating the immune-related genes and various functional genes. The structure, expression and tyrosine-based phosphorylation of STAT6 are conserved from fish to mammal. However, except the sporadic reports from zebra fish, the function of fish STAT6 has not been well reported. Here, we cloned and characterized the full length cDNA sequence of grass carp (Ctenopharyngodon idella) STAT6 (CiSTAT6). Meanwhile, the activation mechanism and the potential function of CiSTAT6 were studied. The full length cDNA of CiSTAT6 is 2747 bp with an ORF of 2313 bp encoding a polypeptide of 770 amino acids. Phylogenetic tree analysis revealed that CiSTAT6 shares the maximum homology with Cyprinus carpio STAT6. CiSTAT6 was significantly up-regulated and interacted with each other to form the homodimer after treatment with poly I:C. The transfected CiSTAT6 in fish cell lines can activate the promoter activities of CCL20 and Bcl-xl and increase their mRNA levels. In addition, we also found that CiSTAT6 can increase cell viability and inhibit cell apoptosis. Taken together, grass carp STAT6 plays an important part in innate immunity and anti-apoptosis.

Ctenopharyngodon idella IRF2 and ATF4 down-regulate the transcriptional level of PRKRA.

PRKRA (interferon-inducible double-stranded RNA-dependent protein kinase activator A) is a protective protein which regulates the adaptation of cells to ER stress and virus-stimulated signaling pathways by activating PKR. In the present study, a grass carp (Ctenopharyngodon idella) PRKRA full-length cDNA (named CiPRKRA, KT891991) was cloned and identified. The full-length cDNA is comprised of a 5' UTR (36 bp), a 3' UTR (350 bp) and the longest ORF (882 bp) encoding a polypeptide of 293 amino acids. The deduced amino acid sequence of CiPRKRA contains three typical dsRNA binding motifs (dsRBM). Phylogenetic tree analysis revealed a closer evolutionary relationship of CiPRKRA with other fish PRKRA, especially with Danio rerio PRKRA. qRT-PCR showed that CiPRKRA was significantly up-regulated after stimulation with tunicamycin (Tm) and Poly I:C in C. idella kidney (CIK) cells. To further study its transcriptional regulation, the partial promoter sequence of CiPRKRA (1463 bp) containing one ISRE and one CARE was cloned by Tail-PCR. Subsequently, grass carp IRF2 (CiIRF2) and ATF4 (CiATF4) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind Resin. In vitro, both CiIRF2 and CiATF4 bound to CiPRKRA promoter with high affinity by gel mobility shift assays, revealing that IRF2 and ATF4 might be potential transcriptional regulatory factors for CiPRKRA. Dual-luciferase reporter assays were applied to further investigate the transcriptional regulation of CiPRKRA in vivo. Recombinant plasmid of pGL3-PRKRAPro was constructed and transiently co-transfected into CIK cells with pcDNA3.1-CiIRF2 and pcDNA3.1-CiATF4, respectively. The results showed that both CiIRF2 and CiATF4 significantly decreased the luciferase activity of pGL3-PRKRAPro, suggesting that they play a negative role in CiPRKRA transcription.

Room temperature removal of high-space-velocity formaldehyde boosted by fixing Pt nanoparticles into Beta zeolite framework.

Catalytic oxidation of volatile organic compounds like formaldehyde (HCHO) over the noble metals catalysts at room temperature is among the most promising strategies to control indoor pollution but remains one challenge to maximize the efficiency of noble metal species. Herein, we demonstrated the straightforward encapsulation of highly dispersive Pt nanoparticles (NPs) within BEA zeolite and adjacent with the surface hydroxyl groups to reach the synergistic HCHO oxidation at 25 °C. High efficiency and long-term stability was reached under large space velocity (∼100% conversion at 180,000 mL (gcat × h)-1 and >95% at 360,000 mL (gcat × h)-1), affording rapid elimination rate of 129.4 µmol (gPt × s)-1 and large turnover frequency of 2.5 × 10-2 s-1. This is the first synergy example derived from the hydroxyl groups and confined noble metals within zeolites that accelerated the rate-determining step, the formate transformation, in the HCHO elimination.

Grass carp STK38 regulates IFN I expression by decreasing the phosphorylation level of GSK3ß.

As a member of NDR protein kinase family and a novel protein kinase of Hippo signal pathway, Serine/threonine kinase 38 (STK38) plays a very significant role in the innate immune. In mammals, STK38 performs its function by combining with GSK3ß. Nowadays, there are few reports of STK38 in fish. In order to explain the function of fish STK38 in the innate immunity, we cloned the ORF of grass carp (Ctenopharyngodon idella) STK38 (CiSTK38) and the related kinase GSK3ß (CiGSK3ß). Phylogenetic trees revealed that CiSTK38 and CiGSK3ß evolved closer kinship with sinocyclocheilus grahami STK38 and siniperca chuatsi GSK3ß respectively. CiSTK38 and CiGSK3ß can respond to the intradermal injection of poly (I:C) in grass carp different tissues and the transfection of poly I:C in CIK cells. Subcellular localization revealed the CiGSK3ß were broadly distributed through the cytoplasm, whereas CiSTK38 were observed both in cytoplasm and nucleus. However, when they were co-transferred into cells, the two proteins were found to aggregate in the nucleus. GST-pulldown and co-immunoprecipitation analysis revealed that CiSTK38 can physically interact with CiGSK3ß. Phos-tag PAGE illustrated CiSTK38 can decrease the phosphorylation and auto-phosphorylation level of CiGSK3ß at Ser9 and at Tyr216. To investigate the functional correlation between CiSTK38 and CiGSK3ß, we overexpressed CiSTK38 and CiGSK3ß in CIK cells and found that they can up-regulate the expression of IFN I. In short, we demonstrated that CiSTK38 can confer CiGSK3ß kinase activity by reducing its phosphorylation level. Result from this study strongly suggested that the anti-viral immune effects elicited by poly (I:C) in part were mediated through activation of CiGSK3ß. The findings provided scientific basis for the anti-viral immune mechanism of STK38 and GSK3ß in fish.

Fish SAMHD1 performs as an activator for IFN expression.

As a host limiting factor, Sterile Alpha Motif and Histidine-Aspartate Domain 1 protein (SAMHD1) is associated with IRF3-mediated antiviral and apoptotic responses in mammals. However, the antiviral mechanism of SAMHD1 remains indistinct in fish. In this study, we found the expression of Ctenopharyngodon idella SAMHD1 (MF326081) was up-regulated after transfection with poly I:C (dsRNA analog), B-DNA or Z-DNA into C. idella kidney cells (CIKs), but these expression profiles had no obvious change when the cells were incubated with these nucleic acids. These data may indicate that CiSAMHD1 participates in the intracellular PRR-mediated signaling pathway rather than extracellular PRR-mediated signaling pathway. Subcellular localization assay suggested that a part of over-expressed CiSAMHD1 were translocated from nuclear to cytoplasm when C. idella ovary cells (COs) were transfected with poly I:C, B-DNA or Z-DNA. Nucleic acid pulldown assays were performed to investigate the reason for nuclear-cytoplasm translocation of CiSAMHD1. The results showed that CiSAMHD1 had a high affinity with B-DNA, Z-DNA and ISD-PS (dsRNA analog). In addition, co-IP assays revealed the interaction of CiSAMHD1 with CiSTING (KF494194). Taken together, all these results suggest that grass carp SAMHD1 performs as an activator for innate immune response through STING-mediated signaling pathway.

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.

Diagnostic test of rifampicin resistance in Mycobacterium tuberculosis: a meta-analysis.

OBJECTIVE: To evaluate available evidence on the diagnostic value of radioactivity-free cultivation and detection technologies for rapid detection of rifampicin resistance in Mycobacterium tuberculosis. METHODS: A fully recursive literature search was conducted in PubMed, EMBASE, Biosis, Web of Science (all 1990-2010), CBMWeb (1978-2010), and Google Scholar. QUADAS items were used to evaluate the quality of included studies. Sensitivity, specificity, Summary receiver-operating curve SEN, SPE, SROC, and related techniques were used to assess the diagnostic value of radioactivity-free Mycobacterium tuberculosis cultivation and detection technologies. RESULTS: Six studies were included in the final analysis. The MB/BacT, BACTEC MGIT 960, and Manual MGIT systems were highly sensitive and specific for detecting rifampicin-resistant TB. The summary SEN and summary SPE of the MB/BacT and BACTEC MGIT 960 systems were 100%, 99%, 100%, and 96%, respectively. The SROC of the BACTEC MGIT 960 system was 0.9943. CONCLUSION: We recommended that the BACTEC 460 system be replaced by MB/BacT or BACTEC MGIT 960 as the final diagnostic test for rifampicin resistance in Mycobacterium tuberculosis. More studies are needed on the diagnostic value of other radioactivity-free cultivation and detection technologies to reliably determine their sensitivity and specificity for this bacterium.