Grass carp Il-2 promotes neutrophil extracellular traps formation via inducing ROS production and autophagy in vitro.

Interleukin (IL)-2 has been reported to regulate neutrophil functions in humans, mice, pigs and chicken although it is a key regulator of T cells. Consistently, we found that grass carp (Ctenopharyngodon idellus) interleukin-2 (gcIl-2) is capable of modulating the antimicrobial activities of neutrophils via regulating granzyme B- and perforin-like gene expression in our previous study. In the present study, stimulation of gcIl-2 on neutrophil extracellular traps (NETs) formation in grass carp neutrophils was demonstrated by detecting free DNA release, histone H3 citrullination and morphological changes of the cells. Further investigation revealed that reactive oxygen species (ROS) production from NADPH oxidase but not mitochondria was involved in NETosis induced by gcIl-2. Aside from ROS, autophagy was disclosed to be indispensable for NETosis induced by gcIl-2. These converging lines of evidence suggested that fish Il-2 could induce NETs formation via NADPH oxidase-derived ROS- and autophagy-dependent pathways in fish species which is evolutionarily conserved with that in mammals. It is noteworthy that these two pathways did not interplay with each other in Il-2-stimulated NETosis. The mechanisms governing autophagy induced by Il-2 were also explored in the present study, showing that Il-2 modulated the action of high mobility group box 1 (HMGB1) protein to stimulate autophagy, leading to NETs formation in fish neutrophils. These results provided a new insight to the function of Il-2 in fish neutrophils, and a clue about the regulation of NETosis in the lower vertebrates.

MXene-Based Aptameric Fluorosensor for Sensitive and Rapid Detection of COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-caused COVID-19 pandemic has rapidly escalated into the largest global health emergency, which pushes to develop detection kits for the detection of COVID-19 with high sensitivity, specificity, and fast analysis. Here, aptamer-functionalized MXene nanosheet is demonstrated as a novel bionanosensor that detects COVID-19. Upon binding to the spike receptor binding domain of SARS-CoV-2, the aptamer probe is released from MXene surface restoring the quenched fluorescence. The performances of the fluorosensor are evaluated using antigen protein, cultured virus, and swab specimens from COVID-19 patients. It is evidenced that this sensor can detect SARS-CoV-2 spike protein at final concentration of 38.9 fg mL-1 and SARS-CoV-2 pseudovirus (limit of detection: 7.2 copies) within 30 min. Its application for clinical samples analysis is also demonstrated successfully. This work offers an effective sensing platform for sensitive and rapid detection of COVID-19 with high specificity.

IFN-γ Manipulates NOD1-Mediated Interaction of Autophagy and Edwardsiella piscicida to Augment Intracellular Clearance in Fish.

Edwardsiella piscicida is an intracellular pathogenic bacterium accounting for significant losses in farmed fish. Currently, cellular and molecular mechanisms underlying E. piscicida-host cross-talk remain obscure. In this study, we revealed that E. piscicida could increase microtubule-associated protein L chain 3 (LC3) puncta in grass carp (Ctenopharyngodon idella) monocytes/macrophages and a carp cell line, Epithelioma papulosum cyprini The autophagic response was confirmed by detecting the colocalization of E. piscicida with LC3-positive autophagosomes and LysoTracker-probed lysosomes in the cells. Moreover, we unveiled the autophagic machinery targeting E. piscicida by which the nucleotide-binding oligomerization domain receptor 1 (NOD1) functioned as an intracellular sensor to interact and recruit autophagy-related gene (ATG) 16L1 to the bacteria. Meanwhile, E. piscicida decreased the mRNA and protein levels of NOD1 and ATG16L1 in an estrogen-related receptor-α-dependent manner, suggesting a possible mechanism for this bacterium escaping autophagy. Subsequently, we examined the effects of various E. piscicida virulence factors on NOD1 expression and found that two of them, EVPC and ESCB, could reduce NOD1 protein expression via ubiquitin-dependent proteasomal degradation. Furthermore, an intrinsic regulator IFN-γ was found to enhance the colocalization of E. piscicida with NOD1 or autophagosomes, suggesting its involvement in the interaction between autophagy and E. piscicida Along this line, a short-time treatment of IFN-γ caused intracellular E. piscicida clearance through an autophagy-dependent mechanism. Collectively, our works demonstrated NOD1-mediated autophagy-E. piscicida dialogues and uncovered the molecular mechanism involving autophagy against intracellular bacteria in fish.

Regulation of Il-2 on the expression of granzyme B- and perforin-like genes and its functional implication in grass carp peripheral blood neutrophils.

Granzyme (Gzm) B and perforin, both as cytotoxic proteins, can collaborate to induce the death of target cells as well as the microbes. They were originally discovered in cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells and confer the cytotoxic activities of these cells. In the present study, the coding sequences of a granzyme b-like (gcgzmbl) and a perforin-like (gcprfl) genes were cloned from grass carp (Ctenopharyngodon idellus) and their specific antibodies were subsequently prepared and validated. The mRNA and protein expression of these two cytotoxic proteins in grass carp peripheral blood neutrophils was demonstrated by quantitative PCR (qPCR) and immunofluorescence staining, respectively. In the same cell model, expression of gcGzmbl and gcPrfl was stimulated by grass carp interleukin (Il)-2 in a dose- and time-dependent manners and Erk, NF-κB and Stat5 pathways were found to be involved in the regulation of Il-2 on the genes' expression. Additionally, glycolysis was proved to play a role in the stimulation of Il-2 on gcGzmbl and gcPrfl expression in peripheral blood neutrophils. As combating the invading microorganisms is one of the main functions of neutrophils, the roles of gcGzmbl and gcPrfl in the anti-bacterial activities of grass carp peripheral blood neutrophils were explored. Results showed that immunoneutralization of gcGzmbl or gcPrfl significantly attenuated the antimicrobial abilities of the neutrophils enhanced by Il-2. These findings shed a light on the expression, regulation and functions of granzyme B- and perforin-like proteins in fish peripheral blood neutrophils and enrich the understanding of Il-2 function in fish innate immunity.

OGG1 contributes to hepatocellular carcinoma by promoting cell cycle-related protein expression and enhancing DNA oxidative damage repair in tumor cells.

BACKGROUND: This study aimed to analyze the expression of 8-oxoguanine DNA glycosylase (OGG1) in patients with hepatocellular carcinoma (HCC) and its effect on prognosis by bioinformatics techniques and to determine its possible carcinogenic mechanism through data mining. METHODS: The difference in OGG1 expression between healthy people and HCC patients was searched and analyzed by TCGA and GEO databases, and the effect of OGG1 on prognosis was judged by survival analysis. Meanwhile, the possible molecular mechanism of OGG1 in the tumorigenesis and development of HCC was explored by GO analysis, KEGG analysis, immune infiltration analysis, protein-protein interaction network, promoter methylation analysis, and so forth. Quantitative polymerase chain reaction (qPCR) was used to examine the gene expression in 36 pairs of HCC tissues and adjacent tissues. RESULTS: The expression of OGG1 in HCC patients was higher than that in healthy people, and the overexpression of OGG1 might stimulate cell proliferation by increasing the activity of cell cycle-related proteins. CONCLUSION: The alteration of OGG1 was significantly correlated with the tumorigenesis and development of HCC. OGG1 is expected to be a new biomarker for evaluating the prognosis of HCC and a new target for the treatment of HCC.

Functional characterization of grass carp (Ctenopharyngodon idella) interleukin-2 in head kidney leukocytes.

Interleukin (IL)-2 belongs to the four-helix bundle cytokine family and plays key roles in growth, survival, activation-induced cell death and differentiation of the immune cells. In cyprinid fish, only common carp interleukin-2 (il2) has been cloned because of relatively low sequence homology between carp Il-2 and its homologs in other fish species. In the present study, the coding sequence of grass carp Il-2 (gcIl-2) was cloned and its identity was verified via bioinformatic analysis. Tissue distribution study showed that grass carp il2 (gcil2) mRNA was expressed in thymus, head kidney and gill with relatively high levels. Recombinant gcIl-2 (rgcIl-2) protein was subsequently prepared by using a prokaryotic expression system followed by a refolding method. The purified rgcIl-2 displayed an ability to stimulate the cell proliferation along with an increased mRNA expression of cd4l but not cd8a, igm or mcsfr in grass carp head kidney leukocytes (HKLs), suggesting the possible involvement of gcIl-2 in T helper (Th) cell proliferation. In the same cell model, rgcIl-2 significantly enhanced mRNA expression of some cytotoxic molecules including perforin-like protein 2, granzyme B-like and Fas ligand, indicating the modulation of cytotoxic cells by gcIl-2 in grass carp HKLs. Besides, gene expression of regulatory T (Treg) cell- and Th1/2 cell-related cytokines or transcription factors was detected in grass carp HKLs treated by rgcIl-2. Results showed that rgcIL2 treatment increased the mRNA expression of foxp3, cd25l, ifng2, il12p35, tbet, tnfa, il2, il4/13a, il4/13b and gata3l in HKLs, implying the regulatory roles of Il-2 in the expression of these immune genes and its possible involvement in differentiation of Treg and Th1/2 cells. These observations together with the related studies in other fishes suggest the existence of cytotoxic cells, Treg and Th1/2 subpopulations in fish species and the functional roles of Il-2 in these cells.

NF-κB p65 promotes ovarian cancer cell proliferation and migration via regulating mortalin.

Previous studies show that mortalin, a HSP70 family member, contributes to the development and progression of ovarian cancer. However, details of the transcriptional regulation of mortalin remain unknown. We aimed to determine whether NF-κB p65 participates in the regulation of mortalin expression in ovarian cancer cells and to elucidate the underlying mechanism. Chromatin immunoprecipitation and luciferase reporter assay were used to identify mortalin gene sequences, to which NF-κB p65 binds. Results indicated that NF-κB p65 binds to the mortalin promoter at a site with the sequence 'CGGGGTTTCA'. Using lentiviral pLVX-NF-κB-puro and Lentivirus-delivered NF-κB short hairpin RNA (shRNA), we created ovarian cancer cell lines in which NF-κB p65 was stably up-regulated and down-regulated. Using these cells, we found that downregulation of NF-κB p65 inhibits the growth and migration of ovarian cancer cells. Further experimental evidence indicated that downregulation of NF-κB p65 reduced mortalin, and upregulation of mortalin rescued the proliferation and migration of ovarian cancer cells reduced by NF-κB p65 knockdown. In conclusion, NF-κB p65 binds to the mortalin promoter and promotes ovarian cancer cells proliferation and migration via regulating mortalin.

Molecular characterization of grass carp interleukin-6 receptor and the agonistic activity of its soluble form in head kidney leucocytes.

Interleukin (IL)-6 receptor (IL-6R) can specifically bind to IL-6 and the complex subsequently recruits a transmembrane signal transducer, gp130, to trigger the intracellular signal transduction. IL-6R exists in two forms, a transmembrane IL-6R and a soluble IL-6R (sIL-6R), leading to different signal transduction mechanisms as classic signaling and trans-signaling, respectively. There is now a general consensus that these two modes of signal transduction can mediate anti-inflammatory and pro-inflammatory activities of IL-6. The study on Il-6r is limited although Il-6 has been well studied in teleost. In the present study, a cDNA encoding grass carp Il-6r (gcIl-6r) was isolated. An in-silico analysis showed that gcIl-6r shared the same functional domains and conserved gene synteny at its loci with mouse homologue, and its amino acid sequence was conserved in fish species. A tissue distribution assay demonstrated that gcil6r mRNA was expressed with high levels in immune tissues including spleen and head kidney, and its expression was induced by LPS and Poly I:C in grass carp head kidney leucocytes (HKLs). An in vitro binding assay showed that recombinant soluble gcIl-6r (rgcsIl-6r) could specifically bind to recombinant gcIl-6 (rgcIl-6) protein. Moreover, rgcIl-6 stimulated suppressor of cytokine signaling 3 (socs3)'s mRNA expression in grass carp HKLs and it combined with rgcsIl-6r increased socs3 mRNA expression in CIK cells with gp130 but without Il-6r expression. In HKLs, rgcIl-6 stimulated the mRNA levels of both pro-inflammatory (tnfa and il1b) and anti-inflammatory (il10) cytokines, and rgcsIl-6r could augment these stimulatory effects of gcIl-6. Taken these data together, gcsIl-6r can mediate the immuno-regulatory functions of gcIl-6 and has an agonistic property in these actions of Il-6 in grass carp.

Functional characterization of three fish-specific interleukin-23 isoforms as regulators of Th17 signature cytokine expression in grass carp head kidney leukocytes.

Mammalian Interleukin (IL)-23 is a heterodimeric cytokine with an IL-23-specific P19 subunit and a P40 subunit shared with IL-12, and plays a key role in the regulation of cell differentiation as well as inflammation. We previously demonstrated the existence of three soluble fish Interleukin (Il)-23 isoforms consist of a single P19 and one of three P40 isoforms (P40a/b/c) in grass carp. In the present study, three recombinant grass carp Il-23 (rgcIl-23) isoforms were prepared by linking gcP19 and gcP40a/b/c in a prokaryotic expression system, and then their functional properties were verified in grass carp head kidney leukocytes (HKLs). All three rgcIl-23 isoforms showed the bioactivities to divergently upregulate the mRNA expression of Th17 signature cytokines (il17a/f1, il21, il22 and il26) as well as Il-23 receptor (il23r) in HKLs. Moreover, they also promoted gcIl-17a/f1 secretion in a dose-dependent manner, strengthening their roles in Th17-like response. Furthermore, induction of il17a/f1 and il23r transcription by rgcIl-23 was blocked by a STAT3 inhibitor in grass carp HKLs, suggesting the involvement of STAT3 signaling in these inductions. Taken together, we for the first time identified the bioactivities of fish Il-23 isoforms and particularly revealed the existence of Il-23/Il-17a/f1 axis in fish, thereby advancing our understanding of Th17-like responses in fish immunity.

Identification of an intercellular cell adhesion molecule-1 homologue from grass carp: Evidence for its involvement in the immune cell adhesion in teleost.

Intercellular cell adhesion molecule-1 (ICAM-1) is a single-chain transmembrane glycoprotein which plays key roles in transendothelial migration of leukocytes and interaction between antigen presenting cells and T cells. In teleost, information of cell adhesion-related molecules is still lacking. In this study, we identified a gene from grass carp sharing similar exon and intron organization with human ICAM-1. Cloning and in silico analysis of its homologues in zebrafish and other two cyprinid fishes, respectively demonstrated the existence of the gene in these fishes. Moreover, the molecular features of these genes in fishes were conserved compared with human ICAM-1. In grass carp, the transcripts of this gene were detected with high levels in heart and liver and its mRNA expression in headkidney leukocytes was induced by Il-1ß. Overexpression of this molecule in COS-7 cells could increase the adhesion of the cells with grass carp peripheral blood lymphocytes (PBLs), and the adhesion was further enhanced by lipopolysaccharide stimulation on PBLs. Further studies revealed that the mRNA levels of lymphocyte function-associated antigen-1, a ligand for ICAM-1, were much higher in the PBLs adhering to the COS-7 cells with overexpressing this molecule than in the PBLs alone. These results collectively showed that the newly cloned cDNA encodes grass carp intercellular cell adhesion molecule-1 (Icam-1) and it can mediate the adhesion of PBLs. This provides functional evidence for the existence of Icam-1 in teleost and will facilitate investigation on the transendothelial migration of leukocytes in fish species.

In vitro characterization of grass carp (Ctenopharyngodon idella) IL-26 in regulating inflammatory factors.

Interleukin 26 (IL-26) gene has been identified in human, amphibian and teleost but not in rodents. It is well accepted that IL-26 was a crucial member of IL-10 family which acts as a pro-inflammatory cytokine in human. However, the role of IL-26 in regulating inflammation in lower vertebrates including teleost has not been defined yet. In the present study, grass carp IL-26 (gcIL-26) coding sequence was isolated and identified. Its chromosomal synteny was also analyzed, showing that gcIL-26 gene is flanked by IL-22 and IFN-γ genes with the same transcriptional orientation as seen in human, amphibian and zebrafish. Given that zebrafish and grass carp IL-26 shared relatively low amino acid identities with human IL-26, the functional roles of fish IL-26 are indispensable to be elucidated. Accordingly, recombinant gcIL-26 (rgcIL-26) was prepared by using Pichia pastoris expression system, and it was found to be partially glycosylated. Using grass carp head kidney leucocytes as cell model, rgcIL-26 displayed the bioactivity to stimulate the mRNA expression of some pro-inflammatory cytokines including IL-8, IL-1ß and IL-6, while inhibit mRNA expression of an anti-inflammatory cytokine, IL-10. Moreover, rgcIL-26 also up-regulated inos expression and NO production in grass carp monocytes/macrophages, strengthening its pro-inflammatory properties in fish. Those results collectively demonstrated the functional role of IL-26 in regulating inflammatory response in fish.

Thymosin From Bombyx mori Is Down-Regulated in Expression by BmNPV Exhibiting Antiviral Activity.

Thymosins have been highly conserved during evolution. These hormones exist in many animal species and play an essential role in many biological events. However, little is known regarding the physiological function of silkworm Bombyx mori thymosin (BmTHY). In this study, we investigated the expression pattern of BmTHY in a Bombyx mori larval ovarian cell line (BmN) challenged with Bombyx mori nuclear polyhydrosis virus (BmNPV) and the antiviral effect of recombinant BmTHY (rBmTHY) for Bombyx mori against BmNPV. Western-blot assay and qRT-PCR analysis revealed that the level of BmTHY protein expression and transcription decreased over time when BmN cells were infected by BmNPV. Treatment with endotoxin-free rBmTHY led to a significant reduction in viral titer in the supernatant of BmN cells challenged with BmNPV. The results from antiviral tests performed in vitro and in vivo showed that endotoxin-free rBmTHY improved the survival rate of Bombyx mori infected with BmNPV. These findings suggest that BmTHY exerts immunomodulatory effects on Bombyx mori, rendering them resistant to viral infection.

Chitosan-zinc chelate improves intestinal structure and mucosal function and decreases apoptosis in ileal mucosal epithelial cells in weaned pigs.

The present study was conducted to investigate the effects of chitosan (CS)-Zn on intestinal morphology, mucosal epithelial cell apoptosis and mucosal immune function in weanling pigs. A total of 150 weanling barrows with a body weight of 7.2 kg were randomly allocated into five groups. A basal diet without Zn supplementation was used as the control and other four groups were fed the control diet supplemented with 50 or 100 mg/kg of Zn as CS-Zn, 100 mg/kg of Zn as ZnSO4 and 3000 mg/kg of Zn as ZnO, respectively. The feeding trial lasted for 28 d. The results showed that serum diamine oxidase activities, d-lactate levels and endotoxin contents were lower in pigs fed dietary 100 mg/kg of Zn as CS-Zn or 3000 mg/kg of Zn as ZnO than in pigs fed the control or 100 mg Zn/kg as ZnSO4 diet. The ratios of the villus height:crypt depth of the duodenum, jejunum and ileum were higher in pigs that received 100 mg/kg of Zn as CS-Zn or a high level of Zn as ZnO than in pigs fed the control diet. Moreover, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL)-stained ileal epithelial cells were found in the control group, and apoptotic cells did not appear prominently in pigs that received the 100 mg/kg of CS-Zn or ZnO diet. Secretory IgA concentration in ileal mucus was increased in the dietary group that received 100 mg/kg of CS-Zn or ZnO. These results indicated that dietary 100 mg CS-Zn/kg had similar biological effects to dietary 3000 mg ZnO/kg on intestinal morphology, mucosal epithelial cell apoptosis and mucosal immune function.

Network Pharmacology Combined with an Experimental Validation Study to Reveal the Effect and Mechanism of Eucommia ulmoides Leaf Polysaccharide against Immunomodulation.

In the present study, the immuno-enhancing effect of Eucommia ulmoides leaf polysaccharide (ELP) was investigated in immunosuppressed mice induced by cyclophosphamide (CTX). To evaluate the immune enhancement mechanism of ELP, the immunoregulation effect of ELP was evaluated in vitro and in vivo. ELP is primarily composed of arabinose (26.61%), galacturonic acid (25.1%), galactose (19.35%), rhamnose (16.13%), and a small amount of glucose (12.9%). At 1000~5000 µg·mL-1, ELP could significantly enhance the proliferation and the phagocytosis of macrophages in vitro. Additionally, ELP could protect immune organs, reduce pathological damage, and reverse the decrease in the hematological indices. Moreover, ELP significantly increased the phagocytic index, enhanced the ear swelling response, augmented the production of inflammatory cytokines, and markedly up-regulated the expression of IL-1ß, IL-6, and TNF-α mRNA levels. Furthermore, ELP improved phosphorylated p38, ERK1/2, and JNK levels, suggesting that MAPKs might be involved in immunomodulatory effects. The results provide a theoretical foundation for exploring the immune modulation function of ELP as a functional food.

Neuropeptide Y-Mediated Gut Microbiota Alterations Aggravate Postmenopausal Osteoporosis.

Postmenopausal osteoporosis (PMO) is often accompanied by neuroendocrine changes in the hypothalamus, which closely associates with the microbial diversity, community composition, and intestinal metabolites of gut microbiota (GM). With the emerging role of GM in bone metabolism, a potential neuroendocrine signal neuropeptide Y (NPY) mediated brain-gut-bone axis has come to light. Herein, it is reported that exogenous overexpression of NPY reduced bone formation, damaged bone microstructure, and up-regulated the expressions of pyroptosis-related proteins in subchondral cancellous bone in ovariectomized (OVX) rats, but Y1 receptor antagonist (Y1Ra) reversed these changes. In addition, it is found that exogenous overexpression of NPY aggravated colonic inflammation, impaired intestinal barrier integrity, enhanced intestinal permeability, and increased serum lipopolysaccharide (LPS) in OVX rats, and Y1Ra also reversed these changes. Most importantly, NPY and Y1Ra modulated the microbial diversity and changed the community composition of GM in OVX rats, and thereby affecting the metabolites of GM (e.g., LPS) entering the blood circulation. Moreover, fecal microbiota transplantation further testified the effect of NPY-mediated GM changes on bone. In vitro, LPS induced pyroptosis, reduced viability, and inhibited differentiation of osteoblasts. The study demonstrated the existence of NPY-mediated brain-gut-bone axis and it might be a novel emerging target to treat PMO.

Expression analysis of grass carp Foxp3 and its biologic effects on CXCL-8 transcription in non-lymphoid cells.

Teleost Forkhead box protein P3 (Foxp3) expression was discovered not only in regulatory T cells (Tregs) but also in other cells. Compared to the extensive study on its roles in lymphoid cells, the expression pattern and biological roles of Foxp3 in non-lymphoid cells have not been elucidated in both mammals and fish species. In the present study, grass carp Foxp3 (gcFoxp3) mRNA expression was detected in different cell types, showing that it has a moderate expression level in peripheral blood leukocytes (PBLs), head kidney leukocytes (HKLs) and grass carp fibroblast-like kidney cells (CIK cells). Interestingly, gcFoxp3 mRNA and protein expression could be significantly stimulated by polyinosinic-polycytidylic acid (poly I:C) in CIK cells, indicating its participation in poly I:C-induced immune response in non-lymphoid cells. To further investigate the function of gcFoxp3, its overexpression plasmid was constructed and transfected into CIK cells. After 24 h of transfection, grass carp C-X-C chemokine ligand (CXCL) 8 (gcCXCL-8) mRNA expression was elevated, implying the modulatory role of gcFoxp3 in gcCXCL-8 mRNA expression. This notion was further supported by the features of gcCXCL-8 promoter which contained a putative Foxp3 binding site at -2196 to -2190 region. Poly I:C or overexpression of gcFoxp3 obviously stimulated gcCXCL-8 promoter activity and deletion of gcFoxp3 binding region on the promoter abolished this stimulation, revealing that Foxp3 is pivotal for transcription of CXCL-8 induced by poly I:C. In conclusion, our results collectively demonstrate expression pattern of teleost Foxp3, and illuminate novel immune function of fish Foxp3 in regulating chemokine transcription in non-lymphoid cells.

Identification and functional characterization of interleukin-12 receptor beta 1 and 2 in grass carp (Ctenopharyngodon idella).

Interleukin 12 (IL-12) binds its receptor complex of IL-12 receptor beta 1 (IL-12Rß1) and IL-12Rß2 to transduce cellular signaling in mammals. In teleosts, the function of Il-12 is drawing increasing attention, but molecular and functional features of Il-12 receptors remain obscure. Especially, the existence of multiple Il-12 isoforms in some fish species elicits the requirement to clarify their receptors. In this study, we isolated three cDNA sequences as Il-12 receptor candidates from grass carp, entitled as grass carp Il-12rß1 (gcIl-12rß1), gcIl-12rß2a and gcIl-12rß2b. In silico analysis showed that gcIl-12rß1 and gcIl-12rß2a shared the conserved gene locus and similar structure characteristics with their orthologues of zebrafish, frog, chicken, mouse and human, respectively. However, the Il-12rß2b of grass carp and zebrafish was similar to IL-27Ra in non-fish species. Further locally installed BLAST and gene synteny analysis uncovered three gcIl-12 receptors being single copied genes. Tissue distribution assay revealed that gcil12rß1 and gcil12rß2a transcripts were predominantly expressed in head kidney, differing from the even distribution of gcil12rß2b transcripts in all detected tissues. Subsequently, the binding ability and antagonistic effects of recombinant extracellular region of gcIl-12rß1 with recombinant grass carp Il-12 (rgcIl-12) isoforms were explored, providing functional evidence of the newly cloned gcIl-12rß1 being genuine orthologues of mammalian IL-12Rß1. Moreover, our data showed that gcIl-12rß1 and gcIl-12rß2a but not gcIl-12rß1 and gcIl-12rß2b mediated the effects of rgcIl-12 isoforms on ifn-γ promoter activity, thereby revealing Il-12 receptor signaling in fish. These results identified grass carp Il-12 receptors, thereby advancing our understanding of Il-12 isoform signaling in fish.

Megakaryocytes Mediate Hyperglycemia-Induced Tumor Metastasis.

High blood glucose has long been established as a risk factor for tumor metastasis, yet the molecular mechanisms underlying this association have not been elucidated. Here we describe that hyperglycemia promotes tumor metastasis via increased platelet activity. Administration of glucose, but not fructose, reprogrammed the metabolism of megakaryocytes to indirectly prime platelets into a prometastatic phenotype with increased adherence to tumor cells. In megakaryocytes, a glucose metabolism-related gene array identified the mitochondrial molecular chaperone glucose-regulated protein 75 (GRP75) as a trigger for platelet activation and aggregation by stimulating the Ca2+-PKCα pathway. Genetic depletion of Glut1 in megakaryocytes blocked MYC-induced GRP75 expression. Pharmacologic blockade of platelet GRP75 compromised tumor-induced platelet activation and reduced metastasis. Moreover, in a pilot clinical study, drinking a 5% glucose solution elevated platelet GRP75 expression and activated platelets in healthy volunteers. Platelets from these volunteers promoted tumor metastasis in a platelet-adoptive transfer mouse model. Together, under hyperglycemic conditions, MYC-induced upregulation of GRP75 in megakaryocytes increases platelet activation via the Ca2+-PKCα pathway to promote cancer metastasis, providing a potential new therapeutic target for preventing metastasis. SIGNIFICANCE: This study provides mechanistic insights into a glucose-megakaryocyte-platelet axis that promotes metastasis and proposes an antimetastatic therapeutic approach by targeting the mitochondrial protein GRP75.

An Efficient Bifunctional Electrocatalyst of Phosphorous Carbon Co-doped MOFs.

It is eager to develop high-performance and cheap bifunctional electrochemical catalysts for both of the oxygen reduction reaction (ORR) or oxygen evolution reaction (OER) for the energy crisis and environmental problems. Herein, we report a series of ZIF-derived Co-P-C co-doped polyhedral materials with a well-defined morphology. The optimized catalyst Co/P/MOFs-CNTs-700 exhibited favorable electrochemical activities with the lowest overpotential of 420 mV to achieve the current density of 10 mA cm-2 for OER and the half potential of 0.8 V for ORR in 0.1 M NaOH. The performance can be well improved by doping phosphorous resource which greatly changed its morphology. Meanwhile, the doped carbon resources also improve the conductivity, which makes it a promising bifunctional electrochemical catalyst and can be comparable with the commercial electrocatalysts.

Regulation of Il-10 gene expression by Il-6 via Stat3 in grass carp head kidney leucocytes.

Interleukin (IL)-10 is a critical anti-inflammatory and late cytokine being produced after the proinflammatory mediators while IL-6 is a promptly synthesized cytokine in response to inflammation in mammals. This chronological expression of interleukin (Il)-6 and Il-10 was also found in grass carp head kidney leucocytes (HKLs) treated by heat-killed Aeromonas hydrophila, supporting the possible interplay between grass carp (gc)Il-6 and gcIl-10 in HKLs. Our further findings were in agreement with this hypothesis that recombinant gcIl-6 (rgcIl-6) promptly and transiently increased gcil10 mRNA levels in grass carp HKLs. Moreover, rgcIl-6 enhanced its own mRNA level and this self-enhancement of gcil6 mRNA level could be partially blocked by rgcIl-10. These results collectively suggest that gcIl-10 production stimulated by gcIl-6 may provide a negative feedback to gcIl-6 production. Interestingly, rgcIl-6 significantly decreased gcil10 mRNA levels in grass carp HKLs after the treatment for 12 and 24 h in contrast to its enhancement of gcil10 levels after the treatment for 3 h. Involvement of Stat3 but not MEK, p38 MAPK or JNK pathway in the increase of gcil10 mRNA levels by rgcIl-6 was revealed by using the signaling pathway inhibitors. This was supported by the fact that rgcIl-6 stimulated Stat3 phosphorylation in grass carp HKLs. Furthermore, rgcIl-6 had no effect on the stability of gcil10 mRNA after the treatment for 3 to 36 h while it increased gcil10 promoter activity after the treatment for 24 h. Taken these data together, gcIl-6 can stimulate Il-10 production at early stage but subsequently inhibit il10 mRNA expression in grass carp HKLs, shedding light on the dynamic regulation of il10 mRNA expression by Il-6 in fish immune cells.