Study on Characteristics of Ultrasound-Assisted Fracture Splitting for AISI 1045 Quenched and Tempered Steel.

Ultrasonic vibration-assisted con-rod fracture splitting (UV-CFS) was used to carry out the fracture experiment of 1045 quenched and tempered steel. The effect of ultrasonic vibration on the fracture properties was studied, the fracture microstructure and the evolution of dislocations near the fracture were analyzed and the microscopic mechanism was analyzed. The results show that in the case of conventional fracture splitting without amplitude, the dimple and the fracture belong to ductile fracture. With the increase in ultrasonic amplitude, the plasticity and pore deformation of the con-rod samples decrease at first and then increase; when the amplitude reaches a certain point, the load required for cracking is reduced to a minimum and the ultrasonic hardening effect is dominant, resulting in a decrease in the plasticity of the sample, a cleavage fracture, a brittle fracture, the minimum pore deformation and high cracking quality. The research results also show that with the increase in ultrasonic amplitude, the fracture dislocation density decreases at first, then increases, and dislocation entanglement and grain breakage appear, then decrease, and multiple dislocation slip trajectories appear. The changes in the dislocation density and microstructure are consistent with the above results.

USP14 exhibits high expression levels in hepatocellular carcinoma and plays a crucial role in promoting the growth of liver cancer cells through the HK2/AKT/P62 axis.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common malignant tumor with strong invasiveness and poor prognosis. Previous studies have demonstrated the significant role of USP14 in various solid tumors. However, the role of USP14 in the regulation of HCC development and progression remains unclear. METHODS: We discovered through GEO and TCGA databases that USP14 may play an important role in liver cancer. Using bioinformatics analysis based on the Cancer Genome Atlas (TCGA) database, we screened and identified USP14 as highly expressed in liver cancer. We detected the growth and metastasis of HCC cells promoted by USP14 through clone formation, cell counting kit 8 assay, Transwell assay, and flow cytometry. In addition, we detected the impact of USP14 on the downstream protein kinase B (AKT) and epithelial-mesenchymal transition (EMT) pathways using western blotting. The interaction mechanism between USP14 and HK2 was determined using immunofluorescence and coimmunoprecipitation (CO-IP) experiments. RESULTS: We found that sh-USP14 significantly inhibits the proliferation, invasion, and invasion of liver cancer cells, promoting apoptosis. Further exploration revealed that sh-USP14 significantly inhibited the expression of HK2. Sh-USP14 can significantly inhibit the expression of AKT and EMT signals. Further verification through immunofluorescence and CO-IP experiments revealed that USP14 co-expressed with HK2. Further research has found that USP14 regulates the glycolytic function of liver cancer cells by the deubiquitination of HK2. USP14 regulates the autophagy function of liver cancer cells by regulating the interaction between SQSTM1/P62 and HK2. CONCLUSIONS: Our results indicate that USP14 plays a crucial role in the carcinogenesis of liver cancer. We also revealed the protein connections between USP14, HK2, and P62 and elucidated the potential mechanisms driving cancer development. The USP14/HK2/P62 axis may be a new therapeutic biomarker for the diagnosis and treatment of HCC.

Particle Size-Controlled Oxygen Reduction and Evolution Reaction Nanocatalysts Regulate Ru(bpy)32+'s Dual-potential Electrochemiluminescence for Sandwich Immunoassay.

Multiple signal strategies remarkably improve the accuracy and efficiency of electrochemiluminescence (ECL) immunoassays, but the lack of potential-resolved luminophore pairs and chemical cross talk hinders their development. In this study, we synthesized a series of gold nanoparticles (AuNPs)/reduced graphene oxide (Au/rGO) composites as adjustable oxygen reduction reaction and oxygen evolution reaction catalysts to promote and modulate tris(2,2'-bipyridine) ruthenium(II) (Ru(bpy)32+)'s multisignal luminescence. With the increase in the diameter of AuNPs (3 to 30 nm), their ability to promote Ru(bpy)32+'s anodic ECL was first impaired and then strengthened, and cathodic ECL was first enhanced and then weakened. Au/rGOs with medium-small and medium-large AuNP diameters remarkably increased Ru(bpy)32+'s cathodic and anodic luminescence, respectively. Notably, the stimulation effects of Au/rGOs were superior to those of most existing Ru(bpy)32+ co-reactants. Moreover, we proposed a novel ratiometric immunosensor construction strategy using Ru(bpy)32+'s luminescence promoter rather than luminophores as tags of antibodies to achieve signal resolution. This method avoids signal cross talk between luminophores and their respective co-reactants, which achieved a good linear range of 10-7 to 10-1 ng/ml and a limit of detection of 0.33 fg/ml for detecting carcinoembryonic antigen. This study addresses the previous scarcity of the macromolecular co-reactants of Ru(bpy)32+, broadening its application in biomaterial detection. Furthermore, the systematic clarification of the detailed mechanisms for converting the potential-resolved luminescence of Ru(bpy)32+ could facilitate an in-depth understanding of the ECL process and should inspire new designs of Ru(bpy)32+ luminescence enhancers or applications of Au/rGOs to other luminophores. This work removes some impediments to the development of multisignal ECL biodetection systems and provides vitality into their widespread applications.

Tri-objective optimization of a waste-to-energy plant with super critical carbon dioxide and multi-effect water desalination for building application based on biomass fuels.

In the present research, an innovative biomass-based energy system for the production of electricity and desalinated water for building application is proposed. The main subsystems of this power plant include gasification cycle, gas turbine (GT), supercritical carbon dioxide cycle (s-CO2), two-stage organic Rankine cycle (ORC) and MED water desalination unit with thermal ejector. A comprehensive thermodynamic and thermoeconomic evaluation is performed on the proposed system. For the analysis, first the system is modeled and analyzed from the energy point of view, then it is examined similarly from the exergy point of view, and then an economic analysis (exergy-economic analysis) is performed on the system. Then, we repeat the mentioned cases for several types of biomasses and compare them with each other. Grossman diagram will be presented to better understand the exergy of each point and its destruction in each component of the system. After energy, exergy and economic modeling and analysis, the system is analyzed and modeled using artificial intelligence to help the system optimization process, and the model obtained with genetic algorithm (GA) to maximize the output power of the system, minimize the cost system and maximizing the rate of water desalination is optimized. The basic analysis of the system is analyzed inside the EES software, then it is transferred to the MATLAB software to optimize and check the effect of operational parameters on the thermodynamic performance and the total cost rate (TCR). It is analyzed and modeled artificially and this model is used for optimization. The obtained result will be three-dimensional Pareto front for single-objective and double-objective optimization, for work-output-cost functions and sweetening-cost rate with the specified value of the design parameters. In the single-objective optimization, the maximum work output, the maximum rate of water desalination, and the minimum TCR will be 55,306.89 kW, 17216.86 m3/day, and $0.3760/s, respectively.

Laparoscopic gastric dissociation using a two-port approach in minimally invasive esophagectomy.

BACKGROUND: A new approach for laparoscopic gastric dissociation in minimally invasive esophagectomy (MIE) was attempted. This study aimed to evaluate the short-term outcomes, safety, and efficacy of two-port laparoscopy using the McKeown procedure. METHODS: This retrospective study included 206 consecutive patients with esophageal cancer who underwent a modified two-port laparoscopic or the traditional five-port McKeown procedure at our institution from August 2019 to August 2021. Surgical outcomes of the two methods were compared. RESULTS: Of the 206 patients, 106 (51.46%) underwent the modified two-port procedure, whereas 100 (48.54%) underwent the traditional five-port procedure. Subsequently, 182 propensity score-matched patients were compared. No significant differences were observed in laparoscopic operative time, blood loss during laparoscopic surgery, number of dissected lymph nodes, and pain score on postoperative day 1 between the two groups. The rate of complication and postoperative length of hospital stay did not differ significantly between the two groups. The total hospitalization cost also did not differ significantly between the two groups (p = 0.325). No postoperative deaths occurred in either group. CONCLUSIONS: Our findings demonstrate that laparoscopic gastric dissociation using the two-port approach in MIE is a safe and effective procedure, with short-term outcomes comparable to those of the traditional five-port procedure in patients with esophageal cancer. Larger studies with longer follow-up duration are warranted.

Spectrum-Resolved Electrochemiluminescence to Multiplex the Immunoassay and DNA Probe Assay.

The investigation on electrochemiluminescence (ECL) multiplexing bioassays mainly focuses on simultaneously detecting either proteins or nucleic acids. To overcome the limitation of a short waveband for spectrum-resolved ECL multiplexing bioassays, herein, a highly monochromatic (FWHM <40 nm) and bandgap-engineered ECL luminophore, that is, mercaptopropionic acid-capped and Zn2+-mediated aggregation-induced emission (AIE) assembly of Au nanocrystals (NCs) (Zn2+-AIE-AuNCs), of strong emission and the maximum emission wavelength at 485 nm is developed. The highly monochromatic and bandgap-engineered ECL (485 nm) of Zn2+-AIE-AuNCs can multiplex with the single-waveband and surface-defect-involved ECL (775 nm) of dual-stabilizer-capped CuInS2@ZnS NCs (CIS@ZnS-NCs), enabling a spectrum-resolved ECL multiplexing strategy with different NCs luminophores of a similar particle size as tags. This ECL multiplexing strategy can be utilized to simultaneously detect antigen and DNA probe together without any additional signal amplification procedure and obvious spectroscopic cross-talk, in which the highly monochromatic ECL from Zn2+-AIE-AuNCs is utilized to dynamically determine human carcinoembryonic antigen from 1 pg/mL to 50 ng/mL with a limit of detection (LOD) of 0.3 pg/mL, while the single-waveband ECL from CIS@ZnS-NCs is employed to linearly detect wild-type p53 from 1 pM to 50 nM with a LOD of 0.5 pM. The ECL immunoassay of the proposed strategy is free from the interference of the synchronously conducted DNA probe assay and vice versa, which would open an avenue to couple the immunoassay and DNA probe assay together for clinical colon and breast cancer identification.

Recent advances and future prospects of the potential-resolved strategy in ratiometric, multiplex, and multicolor electrochemiluminescence analysis.

The potential-resolved strategy has gradually demonstrated its distinct values in electrochemiluminescence (ECL) bio-sensing due to its superior characteristics, such as low instrument requirement, short assay time, and improved sample throughput, in conjunction with spatial- and spectrum-resolved techniques. It has recently been widely generalized into versatile multiple-signal ECL analytic platforms, especially in ratiometric and multiplex ECL sensors, in accordance with some specific principles. Furthermore, luminophore pairs with potential- and wavelength-resolved properties have been utilized to visualize biosensors that display multiple colors depending on analyte concentration. However, only a few comprehensive reports on the principles, construction, and application of various ECL sensors in potential-resolved schemes have been published. This review aims to recount the potential-resolved strategy applying to (a) ratiometric ECL sensors, (b) multiplex ECL sensors, and (c) multicolor ECL sensors and to discuss the distinctions and connections among the application principles of these strategies. Finally, the future prospects of ECL-based potential-resolved analysis are explored.

Low-Triggering-Potential Single-Color Electrochemiluminescence from Bovine Serum Albumin-Stabilized Unary Au Nanocrystals for Immunoassays.

Herein, low-triggering-potential (LTP) electrochemiluminescence (ECL) with an onset around 0.0 V (vs Ag/AgCl) is proposed with bovine serum albumin (BSA)-stabilized Au nanocrystals (BSA-AuNCs) as a luminophore and hydrazine hydrate (N2H4) as a coreactant. The BSA-AuNCs/N2H4 system can exhibit efficient LTP-ECL around 0.37 V with the luminophore of both monodispersed and surface-confined states. The LTP-ECL of BSA-AuNCs/N2H4 is a kind of single-color emission with a maximum emission wavelength around 740 nm, which is obviously red-shifted for 80 nm from that of BSA-AuNCs PL, and indicates that the ECL is generated in a surface-defect-involved route instead of the band-gap-engineered route. Importantly, BSA-AuNCs can be utilized as ECL tags to perform sandwich-type immunoassays with acceptable sensitivity and selectivity, which exhibits a wide linear response for determining CA125 from 0.5 to 1000 mU/mL and a limit of detection of 0.05 mU/mL (S/N = 3).

Coreactant-free and Near-Infrared Electrochemiluminescence Immunoassay with n-Type Au Nanocrystals as Luminophores.

The electrochemiluminescence (ECL) bioassay is prominently carried out with the involvement of the coreactant. To remove the detrimental effects of the coreactant on the ECL of luminophores, herein, a promising ECL immunoassay strategy with biocompatible nanoparticles as the luminophore is proposed, which involves directly and electrochemically oxidizing the luminophore methionine-capped Au (Met@Au) nanocrystals (NCs) without the participation of any coreactant. Met@Au NCs are a kind of n-type nanoparticles, and they can be electrochemically injected with valence band (VB) holes around +0.80 and +1.10 V (vs Ag/AgCl). The electrochemically injected exogenous VB hole can recombine with the endogenous conduction band electron of Met@Au NCs and eventually bring out two coreactant-free and near-infrared ECL processes around 0.80 V (ECL-1) and 1.10 V (ECL-2). The intensity of coreactant-free ECL is primarily determined by the electrochemical oxidation-induced hole-injection process. ECL-2 is considerably stronger than ECL-1 and can be exploited for determining the carcinoembryonic antigen (CEA) in a sandwich immunoassay procedure with a linear range from 0.1 to 50 pg/mL as well as a limit of detection of 0.03 pg/mL (S/N = 3).

Evaluation and Analysis of Quantitative Architectural Space Index Based on Analytic Hierarchy Process.

With the continuous development of the social economy, the urban residential structure is also changing, and people have higher and higher requirements for the living environment. Moreover, the landscape construction of public spaces in cities is an important part of the city. It is easy to neglect the comprehensive consideration of historical development and regional culture in architectural projects. The overall lack of individuality in urban design, the lack of characteristics of adapting measures to local conditions, and the blind emphasis on architectural landscaping have led to a serious lack of regional cultural characteristics and spiritual culture in public places. Therefore, in terms of the problems of insufficient landscape construction quality and green concepts being insufficient in the construction environment of cities, an evaluation method system is established in the paper to further study the shortcomings of the scenic area architecture. What is more, relying on the personal experience of the users in the scenic spot and understanding the real needs of the public space landscape environment around the scenic spot, scientific methods and a complete system are applied to make an overall assessment of the public space in the built residential area. Besides, according to the data analysis of the simulation experiment, the advantages and disadvantages of the public space in the residential area are extracted. Combined with the current research status, the green concept design strategy of the public space landscape environment in the scenic spot is summarized. Lastly, according to the data analysis of the simulation experiment, the evaluation satisfaction of the activity atmosphere, plant configuration, and overall layout is improved by 4.2%, 3.7%, and 3.1% compared with other methods, which proves that this study has a more reasonable planning program to meet the various needs of public open space. The development of urban landscape design provides a valuable reference.

Optimization Method for Energy Saving of Rural Architectures in Hot Summer and Cold Winter Areas Based on Artificial Neural Network.

With the phased spatial planning of the rural revitalization strategy, the proportion of architecture energy consumption in the overall social energy consumption is also increasing year by year. Considering the hot summer and cold winter areas, the proportion of architecture energy consumption in the total energy consumption is very large. The ecological environment and natural resources have been greatly threatened, and the issue of energy conservation and environmental protection is imminent. Energy consumption prediction and analysis is an important branch of building energy conservation in the field of building technology and science. Aiming at the energy consumption characteristics of rural architectures in areas with hot summer and cold winter, this paper proposes a method for constructing a neural network model. When building a neural network, the dataset is called and the function is applied randomly to training samples. The data are used for simulation tests to analyze the fit between the predicted results and the calculated results. Flexible forecasting of specific target building energy consumption is achieved, which can provide optimization strategies for updating and adjusting architecture energy efficiency design. The experimental analysis benchmark parameters and the output value in the dataset are compared with the target simulation value. The relative error is less than 4%, and the average relative error value (mean) and the root mean square error (RMSE) value are both controlled within 2%. It is proved that the method in this paper can directly reflect the evaluation of energy consumption by the neural network and realize the high-speed conversion of the generalized model to the concrete goal, which has a certain value and research significance.

Frontier and hot topics in electrochemiluminescence sensing technology based on CiteSpace bibliometric analysis.

Electrochemiluminescence (ECL) is a process in which luminescence is produced by oxidizing or reducing luminophores to transfer radiant charges between electrochemically generated free radicals. Although about 7000 electrochemiluminescence articles have been published in the past 20 years (2000-2021), only a few review articles have summarized the development and application of ECL. In order to better understand the development status, research hotspots and future development trends of ECL technology, it is very necessary to conduct a comprehensive retrospective analysis. This review is based on the bibliometric analysis method of CiteSpace software to quantitatively analyze, visually review and comment on the articles published in the field of ECL in the past 20 years. Quantitatively analyze the authors, the institutional and other basic information to understand the basic development status of ECL, and then visually analyze the high-frequency keywords, burst keywords, keyword clusters, etc., to understand each the evolution of the main research hotspots and development directions of the period, and finally a detailed review and analysis of the selected highly cited articles. We particularly emphasized the development needs of electrochemiluminescence technology in improving the performance of ECL sensors, developing materials with excellent ECL performance, innovating and cooperating with other devices, and developing high-speed and high-throughput ECL sensors. We hope to provide new ideas for promoting the industrial development and clinical application of electrochemiluminescence technology.

High SERPINH1 expression predicts poor prognosis in lung adenocarcinoma.

Background: Serpine Protease Inhibitorclade H1 (SERPINH1) is abnormally expressed in a variety of tumor tissues and is linked to the biological processes of tumorigenesis, migration, invasion, and metastasis. SERPINH1 expression and prognosis in malignant tumors, such as gastric, colorectal, and breast cancers, have previously been studied, but the gene has not yet been investigated in lung adenocarcinoma (LUAD) in terms of prognosis and the potential mechanisms of action. Methods: SERPINH1 was identified as an independent prognostic factor for LUAD in The Cancer Genome Atlas (TCGA) cohort and Affiliated Hospital of Nantong University (NTU) cohort (the LUAD data set) by univariate and multivariate Cox regression analyses. Additionally, we performed immunohistochemical staining to analyze the expression of SERPINH1 in LUAD and normal lung tissue. Based on the TCGA database, we analyzed the correlation of this gene with the tumor mutation burden (TMB), tumor microenvironment, immune infiltration, immune checkpoints, and anti-tumor drugs using the R language-related R package. Results: SERPINH1 was highly expressed in LUAD tissue. Kaplan-Meier survival curves in both the TCGA cohort and the NTU cohort showed that the SERPINH1 low-expression group had a higher survival rate than the high-expression group. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of the SERPINH1 co-expressed genes revealed that the gene was associated with the extracellular matrix and cell proliferation and migration. The analysis of SERPINH1 and the TMB revealed a superior survival advantage for patients with high TMB and high SERPINH1 expression, and worse survival for those with low TMB and high SERPINH1 expression. The analysis of the tumor microenvironment (TME) and immune infiltration revealed that the high and low expression of SERPINH1 was associated with different immune infiltration characteristics. The analysis of the immune checkpoints and anti-tumor drugs showed that immunotherapy and anti-neoplastic treatment were more efficacious in the high SERPINH1 expression group than the low SERPINH1 expression group. Conclusions: Using LUAD tissues and clinical samples, we showed that SERPINH1 can be used as a prognostic biomarker for LUAD. Our findings provide a new approach and strategy for the clinical treatment of LUAD patients.

Bile acids as regulatory molecules and potential targets in metabolic diseases.

Bile acids are important hydroxylated steroids that are synthesized in the liver from cholesterol for intestinal absorption of lipids and other fatty-nutrient. They also display remarkable and immense functions such as regulating immune responses, managing the apoptosis of cells, participating in glucose metabolism, and so on. Some bile acids were used for the treatment or prevention of diseases such as gallstones, primary biliary cirrhosis, and colorectal cancer. Meanwhile, the accumulation of toxic bile acids leads to apoptosis, necrosis, and inflammation. Alteration of bile acids metabolism, as well as the gut microbiota that interacted with bile acids, contributes to the pathogenesis of metabolic diseases. Therefore, the purpose of this review is to summarize the current functions and pre-clinical or clinical applications of bile acids, and to further discuss the alteration of bile acids in metabolic disorders as well as the manipulation of bile acids metabolism as potential therapeutic targets.

Evaluation on the separated effect of 13 hemoglobin variants by a new automatic Hba1c analyzer.

OBJECTIVE: To evaluate the effect of a new type of automatic glycated hemoglobin analyzer on the separation of abnormal hemoglobin. METHODS: Samples diagnosed as hemoglobin variants by capillary electrophoresis and gene testing were selected, and HbA1c analyzer was used for separation and detection. RESULTS: A total of 13 hemoglobin variants in 40 samples could be separated from the normal peaks. CONCLUSIONS: The variant mode of hemoglobin HbA1c can identify a variety of hemoglobin variants, and the type of variants can be preliminarily determined according to the retention time and characteristic peak shape of the variants.

Heterogeneity in Trajectories of Systolic Blood Pressure among Young Adults in Qingdao Port Cardiovascular Health Study.

Background: Although increased age is associated with higher systolic blood pressure (SBP) in general, there may be variation across individuals in how SBP changes over time. The goal of this paper is to identify heterogeneity in SBP trajectories among young adults with similar initial values and identify personal characteristics associated with different trajectory patterns. This may have important implications for prevention and prognosis. Methods: A cohort of 12,468 individuals aged 18-35 years in the Qingdao Port Cardiovascular Health Study in China was followed yearly during 2000-2011. Individuals were categorized into three strata according to their baseline SBP: ≤110 mmHg, 111-130 mmHg, and >130 mmHg. Within each stratum, group-based trajectory analyses were conducted to identify distinct SBP trajectory patterns, and their association with sociodemographic and baseline health characteristics was assessed by ordinal logistic regression. Results: Five distinct groups of individuals exhibiting divergent patterns of increasing, stable or decreasing SBP trends were identified within each stratum. This is a first report to identify a subgroup with decreasing trend in SBP. Individuals with more advanced age, having less than high school education, family history of cardiovascular diseases, greater body mass index, greater waist circumference, and hyperlipidemia at baseline were more likely to experience trajectories of higher SBP within each stratum. Conclusions: The diverging trajectories among young adults with similar initial SBP highlight the need for prevention and feasibility of effective blood pressure control, while the identified risk factors may inform targeted interventions.

miR-27a-3p promotes the malignant phenotypes of osteosarcoma by targeting ten-eleven translocation 1.

Osteosarcoma has become one of the most common primary malignant tumors affecting children and adolescents. Although increasing evidence has indicated that microRNAs (miRNAs or miRs) play important roles in the development of osteosarcoma, the expression of miR­27a­3p and its effects on osteosarcoma are not yet fully understood. In the present study, our data demonstrated that the expression of miR­27a­3p in osteosarcoma cell lines was significantly higher than that in the normal human osteoblastic cell line, hFOB 1.19 cell (P<0.01). In order to explore the role of miR­27a­3p in the development and progression of osteosarcoma, the expression of miR­27a­3p was inhibited by transfection of the MG-63 cells with miR­27a­3p inhibitor. The results revealed that the cell proliferative ability significantly decreased (P<0.01), the number of apoptotic cells significantly increased (P<0.01) and the number of cells passing through the Transwell membrane was significantly reduced in the group transfected with the miR­27a­3p inhibitor (P<0.01). At the same time, the expression of E-cadherin and α-catenin was significantly upregulated (P<0.01), while the expression of vimentin was significantly downregulated in the group transfected with the miR­27a­3p inhibitor (P<0.01). Our results also revealed that the mRNA expression of ten-eleven translocation 1 (TET1) in the osteosarcoma cells was significantly downregulated compared with that in the hFOB 1.19 cells (P<0.01). Luciferase reporter system analysis indicated that miR­27a­3p recognized the TET1 3'-UTR. The protein expression of TET1 significantly increased in the group transfected with the miR­27a­3p inhibitor. The results from CCK-8 assay, flow cytometric assay and Transwell invasion analysis revealed that TET1 knockdown inhibited the biological effects induced by the downregulation of miR­27a­3p. Taken together, the findings of this study indicate that miR­27a­3p is upregulated, while TET1 is downregulated in human osteosarcoma cells. miR­27a­3p inhibition suppresses the proliferation and invasion of osteosarcoma cells, and promotes cell apoptosis via the negative regulation of TET1. miR­27a­3p/TET1 may thus be a potential target for the treatment of osteosarcoma.

Endoplasmic Reticulum Transmembrane Proteins ZDHHC1 and STING Both Act as Direct Adaptors for IRF3 Activation in Teleost.

IFN regulatory factor (IRF)3 is a central regulator for IFN-ß expression in different types of pathogenic infections. Mammals have various pathogenic sensors that are involved in monitoring pathogen intrusions. These sensors can trigger IRF3-mediated antiviral responses through different pathways. Endoplasmic reticulum-associated proteins stimulator of IFN gene (STING) and zinc finger DHHC-type containing 1 (ZDHHC1) are critical mediators of IRF3 activation in response to viral DNA infections. In this study, grass carp STING and ZDHHC1 were found to have some similar molecular features and subcellular localization, and both were upregulated upon stimulation with polyinosinic:polycytidylic acid, B-DNA, or Z-DNA. Based on these results, we suggest that grass carp STING and ZDHHC1 might possess some properties similar to their mammalian counterparts. Overexpression of ZDHHC1 and STING in Ctenopharyngodon idella kidney cells upregulated IFN expression, whereas knockdown of IRF3 inhibited IFN activation. In addition, coimmunoprecipitation and GST pull-down assays demonstrated that STING and ZDHHC1 can interact separately with IRF3 and promote the dimerization and nuclear translocation of IRF3. Furthermore, we also found that small interfering RNA-mediated knockdown of STING could inhibit the expression of IFN and ZDHHC1 in fish cells. Similarly, knockdown of STING resulted in inhibition of the IFN promoter. In contrast, ZDHHC1 knockdown also inhibited IFN expression but had no apparent effect on STING, which indicates that STING is necessary for IFN activation through ZDHHC1. In conclusion, STING and ZDHHC1 in fish can respond to viral DNA or RNA molecules in cytoplasm, as well as activate IRF3 and, eventually, trigger IFN expression.

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.

Poly I:C facilitates the phosphorylation of Ctenopharyngodon idellus type I IFN receptor subunits and JAK kinase.

Members of the Janus kinase (JAK) family, JAK1 and TYK2 take part in JAK-STAT signaling pathway mediated by interferon in mammalian cells. Similar to the mammalian counterparts, fish JAK1 and TYK2 also perform their potential biological activities by phosphorylating cytokine receptors and STAT. In the present study, Ctenopharyngodon idellus JAK1 (CiJAK1) and TYK2 (CiTYK2) were cloned and identified. The full-length cDNA of CiJAK1 (KT724352.1) is 3829 bp, with an Open Reading Frame (ORF) of 3465 bp encoding a putative protein of 1154 amino acids. The full-length cDNA of CiTYK2 (KT724353.1) is 4337 bp, including an ORF of 3168 bp encoding 1055 amino acids. Structurally, both of them have B41, SH2, TyrKc and TyrKc common domains. CiJAK1 and CiTYK2 share a high degree of homology with their respective counterparts from Danio rerio and Cyprinus carpio by phylogenetic tree analysis. Polyinosinic-polycytidylic acid (Poly I:C), a synthetic dsRNA analogue, can launch the JAK-STAT antiviral signaling pathway. To elucidate the molecular mechanism of Poly I:C initiating the antiviral signaling pathway in fish, C. idellus kidney (CIK) cells were stimulated with Poly I:C and then the cell lysates were separated on 10% SDS-PAGE. The results showed that not only Poly I:C drastically increased the expression level of CiJAK1 and CiTYK2, but also it induced the phosphorylation of CiJAK1 and CiTYK2, as well as C. idellus type I IFN receptor subunits, CiCRFB1 and CiCRFB5. In detail, the levels of p-CiJAK1 and p-CiTYK2 were evidently up-regulated at 3 h post stimulation; however the phosphorylation levels of CiCRFB1 and CiCRFB5 displayed a sharp up-regulation at 12 h post stimulation of Poly I:C. As a basic mechnism of feedback regulation of JAK-STAT signaling pathway, overexpression of CiCRFB1 and CiCRFB5 in CIK cells facilitated the phosphorylation of CiJAK1 and CiTYK2.