[Comparison on the diversity of antibiotic resistance genes of three rodent species]. / 3ç§å•®é½¿åŠ¨ç‰©æŠ—ç”Ÿç´ æŠ—æ€§åŸºå› å¤šæ ·æ€§æ¯”è¾ƒ.

Antibiotic resistance genes (ARGs) have attracted widespread attention as a new global pollutant, mainly due to the abuse of antibiotics. To investigate the diversity of ARGs in three rodent species, we used metagenomic sequencing analysis to analyze the diversity of antibiotic resistance genes of 17 individuals of Apodemus peninsulae and 17 individuals of Myodes rufocanus collected from Mudanfeng, and nine individuals of Apodemus agrarius collected from Sandaoguan. A total of 19 types and 248 subclasses of ARGs were detected in the three rodent species. Seven ARGs showed significant difference and five ARGs showed extremely significant difference between M. rufocanus and A. agrarius. Seven ARGs showed significant difference and four ARGs showed extremely significant difference between A. peninsulae and A. agrarius. Four ARGs showed significant difference and five ARGs showed extremely significant difference between M. rufocanus and A. peninsulae. ARGs showing high abundance in three rodents were macrolides, lincoamides, tetracyclines, and ß-lactams. ARGs were widely distributed in the three rodent species. The significant differences in ARGs among different species might be due to the different distribution areas and their diet differentiation. The study could provide a basis for further studies of ARGs in mice and improve the understanding of the harm of ARGs transmission.

Grass carp superoxide dismutases exert antioxidant function and inhibit autophagy to promote grass carp reovirus (GCRV) replication.

Superoxide dismutases (SODs) are potent antioxidants crucial for neutralizing reactive oxygen species (ROS) and protecting organisms from oxidative damage. In this study, we successfully cloned and analyzed two SOD genes, CiSOD1 and CiSOD2, from grass carp (Ctenopharyngodon idellus). CiSOD1 consists of two CuZn signature motifs and two conserved cysteine residues, while CiSOD2 contains a single Mn signature motif. The expression of CiSODs was found to be ubiquitous across all examined tissues, with their expression levels significantly altered after stimulation by grass carp reovirus (GCRV) or pathogen-associated molecular patterns (PAMPs). CiSOD1 was observed to be uniformly distributed in the cytoplasm, whereas CiSOD2 localized in the mitochondria. Escherichia coli transformed with both CiSODs demonstrated enhanced host resistance to H2O2 and heavy metals. Additionally, purified recombinant CiSOD proteins effectively protected DNA against oxidative damage. Furthermore, overexpression of CiSODs in fish cells reduced intracellular ROS, inhibited autophagy, and then resulted in the promotion of GCRV replication. Knockdown of CiSODs showed opposite trends. Notably, these roles of CiSODs in autophagy and GCRV replication were reversed upon treatment with an autophagy inducer. In summary, our findings suggest that grass carp SODs play an important role in decreasing intracellular ROS levels, inhibiting autophagy, and subsequently promoting GCRV replication.

Genome-wide identification of the OMT gene family in Cucumis melo L. and expression analysis under abiotic and biotic stress.

Background: O-methyltransferase (OMT)-mediated O-methylation is a frequent modification that occurs during natural product biosynthesis, and it increases the diversity and stability of secondary metabolites. However, detailed genome-wide identification and expression analyses of OMT gene family members have not been performed in melons. In this study, we aimed to perform the genome-wide identification of OMT gene family members in melon to identify and clarify their actions during stress. Methods: Genome-wide identification of OMT gene family members was performed using data from the melon genome database. The Cucumis melo OMT genes (CmOMTs) were then compared with the genes from two representative monocotyledons and three representative dicotyledons. The basic information, cis-regulatory elements in the promoter, predicted 3-D-structures, and GO enrichment results of the 21 CmOMTs were analyzed. Results: In our study, 21 CmOMTs (named CmOMT1-21) were obtained by analyzing the melon genome. These genes were located on six chromosomes and divided into three groups composed of nine, six, and six CmOMTs based on phylogenetic analysis. Gene structure and motif descriptions were similar within the same classes. Each CmOMT gene contains at least one cis-acting element associated with hormone transport regulation. Analysis of cis-acting elements illustrated the potential role of CmOMTs in developmental regulation and adaptations to various abiotic and biotic stresses. The RNA-seq and quantitative real-time PCR (qRT-PCR) results indicated that NaCl stress significantly induced CmOMT6/9/14/18 and chilling and high temperature and humidity (HTH) stresses significantly upregulated CmOMT14/18. Furthermore, the expression pattern of CmOMT18 may be associated with Fusarium oxysporum f. sp. melonis race 1.2 (FOM1.2) and powdery mildew resistance. Our study tentatively explored the biological functions of CmOMT genes in various stress regulation pathways and provided a conceptual basis for further detailed studies of the molecular mechanisms.

Genome-wide identification and expression analysis of the KCS gene family in soybean (Glycine max) reveal their potential roles in response to abiotic stress.

Very long chain fatty acids (VLCFAs) are fatty acids with chain lengths of 20 or more carbon atoms, which are the building blocks of various lipids that regulate developmental processes and plant stress responses. 3-ketoacyl-CoA synthase encoded by the KCS gene is the key rate-limiting enzyme in VLCFA biosynthesis, but the KCS gene family in soybean (Glycine max) has not been adequately studied thus far. In this study, 31 KCS genes (namely GmKCS1 - GmKCS31) were identified in the soybean genome, which are unevenly distributed on 14 chromosomes. These GmKCS genes could be phylogenetically classified into seven groups. A total of 27 paralogous GmKCS gene pairs were identified with their Ka/Ks ratios indicating that they had undergone purifying selection during soybean genome expansion. Cis-acting element analysis revealed that GmKCS promoters contained multiple hormone- and stress-responsive elements, indicating that GmKCS gene expression levels may be regulated by various developmental and environmental stimuli. Expression profiles derived from RNA-seq data and qRT-PCR experiments indicated that GmKCS genes were diversely expressed in different organs/tissues, and many GmKCS genes were found to be differentially expressed in the leaves under cold, heat, salt, and drought stresses, suggesting their critical role in soybean resistance to abiotic stress. These results provide fundamental information about the soybean KCS genes and will aid in their further functional elucidation and exploitation.

Research progress on the roles of actin-depolymerizing factor in plant stress responses.

Actin-depolymerizing factors (ADFs) are highly conserved small-molecule actin-binding proteins found throughout eukaryotic cells. In land plants, ADFs form a small gene family that displays functional redundancy despite variations among its individual members. ADF can bind to actin monomers or polymerized microfilaments and regulate dynamic changes in the cytoskeletal framework through specialized biochemical activities, such as severing, depolymerizing, and bundling. The involvement of ADFs in modulating the microfilaments' dynamic changes has significant implications for various physiological processes, including plant growth, development, and stress response. The current body of research has greatly advanced our comprehension of the involvement of ADFs in the regulation of plant responses to both biotic and abiotic stresses, particularly with respect to the molecular regulatory mechanisms that govern ADF activity during the transmission of stress signals. Stress has the capacity to directly modify the transcription levels of ADF genes, as well as indirectly regulate their expression through transcription factors such as MYB, C-repeat binding factors, ABF, and 14-3-3 proteins. Furthermore, apart from their role in regulating actin dynamics, ADFs possess the ability to modulate the stress response by influencing downstream genes associated with pathogen resistance and abiotic stress response. This paper provides a comprehensive overview of the current advancements in plant ADF gene research and suggests that the identification of plant ADF family genes across a broader spectrum, thorough analysis of ADF gene regulation in stress resistance of plants, and manipulation of ADF genes through genome-editing techniques to enhance plant stress resistance are crucial avenues for future investigation in this field.

Achieving dual functions of texture modification and water retention of shrimp surimi products with the combination of epigallocatechin-3-gallate and γ-cyclodextrin.

Epigallocatechin-3-gallate (EGCG) exhibits excellent cross-linking effects of myofibrillar proteins, it is prone to self-aggregation, causing excessive cross-linking and moisture loss of gels, which limits its application as a food additive in surimi products. Here, through combination γ-cyclodextrin and EGCG into one inclusion complex, we achieved proper usage of EGCG in shrimp surimi products: elevating both water holding capability and texture properties (hardness, chewiness and resilience). Moreover, the mechanism behind excellent performance was elucidated: as texture modifiers, the complexes improved gel network integrity through intermolecular interactions and moderated disulfide bonds; and as water retainer agents, the complexes promoted transformation of nitrogen in proteins towards the form of protonated amino, facilitating the occurrence of hydration. Furthermore, the inclusion complexes brought a higher phenolic retention within products in contrast with direct addition of EGCG. This work may propose novel insights for the usage of polyphenols as additives in surimi-based products.

Effects of the Entomopathogenic Fungus Mucor hiemalis BO-1 on the Physical Functions and Transcriptional Signatures of Bradysia odoriphaga Larvae.

Mucor hiemalis BO-1 is an entomopathogenic fungus that infects Bradysia odoriphaga, a destructive root maggot. M. hiemalis BO-1 possesses stronger pathogenicity to the larvae than to other stages of B. odoriphaga, and provides satisfactory field control. However, the physiological response of B. odoriphaga larvae to infection and the infection mechanism of M. hiemalis are unknown. We detected some physiological indicators of diseased B. odoriphaga larvae infected by M. hiemalis BO-1. These included changes in consumption, nutrient contents, and digestive and antioxidant enzymes. We performed transcriptome analysis of diseased B. odoriphaga larvae, and found that M. hiemalis BO-1 showed acute toxicity to B. odoriphaga larvae and was as toxic as some chemical pesticides. The food consumption of diseased B. odoriphaga after inoculation with M. hiemalis spores decreased significantly, and there was a significant decrease in total protein, lipid, and carbohydrates in diseased larvae. Key digestive enzymes (protease, α-amylase, lipase, and cellulase) were significantly inhibited during infection. Peroxidase maintained high activity, and the activity of other antioxidant enzymes (catalase, superoxide dismutase, and glutathione S-transferases) first increased and then decreased. Combined with the transcriptional signatures of diseased B. odoriphaga larvae, M. hiemalis BO-1 infection resulted in decreased food consumption, reduced digestive enzyme activity, and altered energy metabolism and material accumulation. Infection was also accompanied by fluctuations in immune function, such as cytochrome P450 and the Toll pathway. Therefore, our results laid a basis for the further study of the interactions between M. hiemalis BO-1 and B. odoriphaga and promoted the genetic improvement of entomopathogenic fungi.

Synthesis and Characterization of Single-Phase α-Cordierite Glass-Ceramics for LTCC Substrates from Tuff.

Single-phase α-cordierite glass-ceramics for a low-temperature co-fired ceramic (LTCC) substrate were fabricated from tuff as the main raw material, using the non-stoichiometric formula of α-cordierite with excess MgO without adding any sintering additives. The sintering/crystallization behavior and the various performances of dielectric properties, thermal expansion, and flexural strength of the glass-ceramics were detected. The results indicated that only single-phase α-cordierite crystal was precipitated from the basic glass sintered at the range 875-950 °C, and µ-cordierite crystal was not observed during the whole sintering-crystallization process. The properties of glass-ceramics were first improved and then deteriorated with the increase in tuff content and sintering temperature. Fortunately, the glass-ceramics sintered at 900 °C with 45 wt.% tuff content possessed excellent properties: high densify (2.62 g∙cm-3), applicable flexural strength (136 MPa), low dielectric loss (0.010, at 10 MHz), low dielectric constant (5.12, at 10 MHz, close to α-cordierite), and suitable coefficients of thermal expansion (CTE, 3.89 × 10-6 K-1).

AtGCS promoter-driven clustered regularly interspaced short palindromic repeats/Cas9 highly efficiently generates homozygous/biallelic mutations in the transformed roots by Agrobacterium rhizogenes-mediated transformation.

Agrobacterium rhizogenes-mediated (ARM) transformation is an efficient and powerful tool to generate transgenic roots to study root-related biology. For loss-of-function studies, transgenic-root-induced indel mutations by CRISPR/Cas9 only with homozygous/biallelic mutagenesis can exhibit mutant phenotype(s) (excluding recessive traits). However, a low frequency of homozygous mutants was produced by a constitutive promoter to drive Cas9 expression. Here, we identified a highly efficient Arabidopsis thaliana gamma-glutamylcysteine synthetase promoter, termed AtGCSpro, with strong activity in the region where the root meristem will initiate and in the whole roots in broad eudicots species. AtGCSpro achieved higher homozygous/biallelic mutation efficiency than the most widely used CaMV 35S promoter in driving Cas9 expression in soybean, Lotus japonicus, and tomato roots. Using the pAtGCSpro-Cas9 system, the average homozygous/biallelic mutation frequency is 1.7-fold and 8.3-fold higher than the p2 × 35Spro-Cas9 system for single and two target site(s) in the genome, respectively. Our results demonstrate the advantage of the pAtGCSpro-Cas9 system used in ARM transformation, especially its great potential in diploids with multiple-copy genes targeted mutations and polyploid plants with multiplex genome editing. AtGCSpro is conservatively active in various eudicots species, suggesting that AtGCSpro might be applied in a wide range of dicots species.

Grass Carp Reovirus Induces Formation of Lipid Droplets as Sites for Its Replication and Assembly.

Grass carp is an important commercial fish in China that is plagued by various diseases, especially the hemorrhagic disease induced by grass carp reovirus (GCRV). Nevertheless, the mechanism by which GCRV hijacks the host metabolism to complete its life cycle is unclear. In this study, we performed lipidomic analysis of grass carp liver samples collected before and after GCRV infection. GCRV infection altered host lipid metabolism and increased de novo fatty acid synthesis. Increased de novo fatty acid synthesis induced accumulation of lipid droplets (LDs). LDs are associated with GCRV viroplasms, as well as viral proteins and double-stranded RNA. Pharmacological inhibition of LD formation led to the disappearance of viroplasms, accompanied by decreased viral replication capacity. Moreover, transmission electron microscopy revealed LDs in close association with the viroplasms and mounted GCRV particles. Collectively, these data suggest that LDs are essential for viroplasm formation and are sites for GCRV replication and assembly. Our results revealed the detailed molecular events of GCRV hijacking host lipid metabolism to benefit its replication and assembly, which may provide new perspective for the prevention and control of GCRV. IMPORTANCE Grass carp reovirus (GCRV) is the most virulent pathogen in the genus Aquareovirus, which belongs to the family Reoviridae. GCRV-induced hemorrhagic disease is a major threat to the grass carp aquaculture industry. Viruses are obligate intracellular parasites that require host cell machinery to complete their life cycle; the mechanism by which GCRV hijacks the host metabolism to benefit viral replication and assembly remains unclear. Our study demonstrated that GCRV infection alters host lipid metabolism and increases de novo fatty acid synthesis. The increased de novo fatty acid synthesis induced accumulation of LDs, which act as sites or scaffolds for GCRV replication and assembly. Our findings illustrate a typical example of how the virus hijacks cellular organelles for replication and assembly and hence may provide new insights for the prevention and control of GCRV.

Grass Carp Prx 3 Elevates Host Antioxidant Activity and Induces Autophagy to Inhibit Grass Carp Reovirus (GCRV) Replication.

Peroxiredoxins are a family of antioxidant proteins that protect cells from oxidative damage caused by reactive oxygen species (ROS). Herein, the peroxiredoxin 3 gene from grass carp (Ctenopharyngodon idellus), named CiPrx3, was cloned and analyzed. The full-length cDNA of CiPrx3 is 1068 bp long, with a 753 bp open reading frame (ORF) that contains a thioredoxin-2 domain, two peroxiredoxin signature motifs, and two highly conserved cysteine residues. CiPrx3 was ubiquitously expressed in all the tested tissues, while its expression level was altered significantly after exposure to grass carp reovirus (GCRV) and pathogen-associated molecular patterns (PAMPs). CiPrx3 was localized in the mitochondria of transfected cells and concentrated in the nucleus after poly (I:C) treatment. Transformation of CiPrx3 into Escherichia coli enhanced host resistance to H2O2 and heavy metals. Purified recombinant CiPrx3 proteins could protect DNA against oxidative damage. Overexpression of CiPrx3 in fish cells reduced intracellular ROS, increased cell viability, and decreased cell apoptosis caused by H2O2 stimulation and GCRV infection. Further study indicated that CiPrx3 induced autophagy to inhibit GCRV replication in fish cells. Collectively, these results imply that grass carp Prx3 elevates host antioxidant activity and induces autophagy to inhibit GCRV replication.

Impact of plastic pollution on outdoor recreation in the existence of bearing capacity and perspective management.

Large-scale increases in plastic waste, greenhouse gas emissions, and fossil fuel depletion all have negative consequences for the environment. Plastic pollution can lead towards negative impacts on outdoor recreational activities. China and the European Union, as world leader in recycling and reuse, are tackling this issue by putting in place measures to counteract this trend for better outdoor recreational activities. As China and EU nations are most attracted by the tourists it is possible that recreational spot can have harmful effects upon wild and human life. So, we analyze the impacts of plastic waste recycling and reuse on outdoor recreation. It is possible to speed up the circular process if industry reduces its resource and energy consumption while also being able to handle plastic waste responsibly, utilize renewable energy sources, generate jobs, and contribute to economic growth, among other things. This research investigates the transition to sustainability in the European Union nations and China between 2000 and 2020 via the prism of resource and energy productivity in the EU nations and China. The Autoregressive Distributed Lag (ARDL) Model, as well as the estimator Driscool Kraay, are employed in this study. There is a statistically significant relationship between plastic recycling and valorization because of plastic pollution leads toward negative impacts on outdoor recreation, as well as resource productivity, according to the data. Increased energy tariffs, insufficient investment in research and development, a lack of job opportunities, and other factors all act as roadblocks to the implementation of circular growth strategies.

Finite element stress analysis of the bearing component and bone resected surfaces for total ankle replacement with different implant material combinations.

BACKGROUND: A proper combination of implant materials for Total Ankle Replacement (TAR) may reduce stress at the bearing component and the resected surfaces of the tibia and talus, thus avoiding implant failure of the bearing component or aseptic loosening at the bone-implant interface. METHODS: A comprehensive finite element foot model implanted with the INBONE II implant system was created and the loading at the second peak of ground reaction force was simulated. Twelve material combinations including four materials for tibial and talar components (Ceramic, CoCrMo, Ti6Al4V, CFR-PEEK) and three materials for bearing components (CFR-PEEK, PEEK, and UHMWPE) were analyzed. Von Mises stress at the top and articular surfaces of the bearing component and the resected surfaces of the tibia and talus were recorded. RESULTS: The stress at both the top and articular surfaces of the bearing component could be greatly reduced with more compliant bearing materials (44.76 to 72.77% difference of peak stress value), and to a lesser extent with more compliant materials for the tibial and talar components (0.94 to 28.09% difference of peak stress value). Peak stresses at both the tibial and talar bone-implant interface could be reduced more strongly by using tibial and talar component materials with smaller material stiffness (7.31 to 66.95% difference of peak stress value) compared with bearing materials with smaller material stiffness (1.11 to 24.77% difference of peak stress value). CONCLUSIONS: Implant components with smaller material stiffness provided a stress reduction at the bearing component and resected surfaces of the tibia and talus. The selection of CFR-PEEK as the material of tibial and talar components and UHMWPE as the material of the bearing component seemed to be a promising material combination for TAR implants. Wear testing and long-term failure analysis of TAR implants with these materials should be included in future studies.

Design and Preliminary Validation of Individual Customized Insole for Adults with Flexible Flatfeet Based on the Plantar Pressure Redistribution.

Flatfoot is a common musculoskeletal deformity. One of the most effective treatments is to wear individually customized plantar pressure-based insoles to help users change the abnormally distributed pressure on the pelma. However, most previous studies were divided only into several plantar areas without detailed plantar characteristic analysis. In this study, a new insole is designed which redistributes pressure following the analysis of characteristic points of plantar pressure, and practical evaluation during walking of subjects while wearing the insole. In total, 10 subjects with flexible flatfeet have participated in the performance of gait experiments by wearing flat insoles, orthotic insoles, and plantar pressure redistribution insoles (PPRI). The results showed that the stance time of PPRI was significantly lower than that of the flat insoles under slow gait. PPRI in the second to third metatarsal and medial heel area showed better unloading capabilities than orthotic insoles. In the metatarsal and heel area, the PPRI also had its advantage in percentage of contact area compared to flat insole and orthotic insole. The results prove that PPRI improves the plantar pressure distribution and gait efficiency of adults with flexible flatfeet, and can be applied into clinical application.

Are The Applications of Tranexamic Acid in Reverse Hybrid Total Knee Arthroplasty (TKA) The Same as Those in Fully Cemented TKA?: A Randomized Controlled Trial.

INTRODUCTION: Traditional fully cemented prosthesis for total knee arthroplasty (TKA) has many disadvantages. Current studies have shown that the effects of mixed fixation TKA are the same as or even better than those of fully cemented TKA. We aimed to compare the total blood loss (TBL) in the two fixation types of TKA and the hemostatic effects of different doses of tranexamic acid (TXA) for reverse hybrid TKA. METHODS: From September 2018 to November 2020, 233 patients with knee osteoarthritis undergoing unilateral TKA were randomly divided into four groups: groups 1 and 2: fully cemented TKA + intra-articular injection (IAI) of either 1 g TXA (n = 54) or 2 g TXA (n = 60); groups 3 and 4: reverse hybrid TKA + IAI of either 1 g TXA (n = 56) or 2 g TXA (n = 63). All patients were administered intravenous drip of TXA (20 mg/kg) as the basic drug. Perioperative and follow-up data of all patients were compared. RESULTS: The TBL in groups 1, 2, and 3 was higher than that in group 4 (P < 0.0001). The TBL in group 1 was significantly less than that in group 3 (P < 0.05). Although there was no significant difference in blood transfusion demand among the four groups (P > 0.05), the number of anemic patients who did not meet the standard of blood transfusion in group 4 decreased significantly (P < 0.0001). There was no significant difference in pain, function or thrombotic complications among all patients. CONCLUSION: The TBL in reverse hybrid TKA is larger than in fully cemented TKA. For reverse hybrid TKA, the hemostatic effect of TXA with 2 g of IAI was significantly better than with 1 g. Although this method does not reduce the need for blood transfusion, it can significantly reduce the incidence of postoperative anemia.

Finite-element analysis of the influence of tibial implant fixation design of total ankle replacement on bone-implant interfacial biomechanical performance.

PURPOSE: Implant loosening in tibia after primary total ankle replacement (TAR) is one of the common postoperative problems in TAR. Innovations in implant structure design may ideally reduce micromotion at the bone-implant interface and enhance the bone-implant fixation and initial stability, thus eventually prevents long-term implant loosening. This study aimed to investigate (1) biomechanical characteristics at the bone-implant interface and (2) the influence of design features, such as radius, height, and length. METHODS: A total of 101 finite-element models were created based on four commercially available implants. The models predicted micromotion at the bone-implant interface, and we investigated the impact of structural parameters, such as radius, length, and height. RESULTS: Our results suggested that stem-type implants generally required the highest volume of bone resection before implantation, while peg-type implants required the lowest. Compared with central fixation features (stem and keel), peripherally distributed geometries (bar and peg) were associated with lower initial micromotions. The initial stability of all types of implant design can be optimized by decreasing fixation size, such as reducing the radius of the bars and pegs and lowering the height. CONCLUSION: Peg-type tibial implant design may be a promising fixation method, which is required with a minimum bone resection volume and yielded minimum micromotion under an extreme axial loading scenario. Present models can serve as a useful platform to build upon to help physicians or engineers when making incremental improvements related to implant design.

Autophagy Inhibits Grass Carp Reovirus (GCRV) Replication and Protects Ctenopharyngodon idella Kidney (CIK) Cells from Excessive Inflammatory Responses after GCRV Infection.

Autophagy is an essential and highly conserved process in mammals, which is critical to maintaining physiological homeostasis, including cell growth, development, repair, and survival. However, the understanding of autophagy in fish virus replication is limited. In this study, we found that grass carp reovirus (GCRV) infection stimulated autophagy in the spleen of grass carp (Ctenopharyngodon idella). Moreover, both Western blot (WB) analysis and fluorescent tracer tests showed that GCRV infection induced the enhancement of autophagy activation in Ctenopharyngodon idella kidney (CIK) cells. Autophagy inducer rapamycin and autophagy inhibitor 3-MA pretreatment can inhibit and promote the proliferation of GCRV, respectively. In addition, grass carp autophagy-related gene 5 (CiATG5)-induced autophagy, as well as rapamycin, showed effects on GCRV replication in CIK cells. Transcriptome analysis revealed that the total number of differentially expressed genes (DEGs) in CiATG5 overexpression groups was less than that of the control during GCRV infection. Enrichment analysis showed that CiATG5 overexpression induced the enhancement of autophagy, lysosome, phagosome, and apoptosis in the early stage of GCRV infection, which led to the clearance of viruses. In the late stage, steroid biosynthesis, DNA replication, terpenoid backbone biosynthesis, and carbon metabolism were upregulated, which contributed to cell survival. Moreover, signaling pathways involved in the immune response and cell death were downregulated in CiATG5 overexpression groups. Further study showed that CiATG5 repressed the expression of inflammatory response genes, including cytokines and type I interferons. Taken together, the results demonstrate that autophagy represses virus replication and attenuates acute inflammatory responses to protect cells.

Characterization and expression of galectin-3 in grass carp (Ctenopharyngodon idella).

Galectins are members of evolutionary conserved lectin family and play important roles in the innate and adaptive immunity of both vertebrates and invertebrates. Galectin-3 is the only chimera galectin with one C-terminal carbohydrate recognition domain (CRD) connected to the N-terminal end. Here, a galectin-3 (named CiGal3) from grass carp was identified and characterized, which encodes polypeptides 362 amino acids with a predicted molecular mass of 36.45 kDa and theoretical isoelectric point of 4.91. The sugar binding motifs involved in carbohydrate binding activity (H-N-R, V-N and W--E-R) were detected in CRD. In comparison to other species, CiGal3 showed the highest similarity and identity to Cyprinus carpio (95.3% sequence similarity and 92.5% sequence identity). The subcellular localization of CiGal3 was distributed in the cytoplasm and nucleus of transfected cells. The CiGal3 transcripts were ubiquitously expressed in all checked tissues and highly expressed in immune tissues. In addition, the expression of CiGal3 in liver and spleen was induced post grass carp reovirus (GCRV), lipopolysaccharide (LPS), and polyinosinic:polycytidylic acid (poly I:C) challenge. These results suggest that CiGal3 plays a vital role in the immune system.

Identification, expression and functional characterisation of CYP1A in grass carp (Ctenopharyngodon idella).

In mammal, CYP1A has attracted special attention due to its important roles in the oxidative metabolism. In fish, the researches on CYP1A are more focus on its roles in pollution in water environments, but the immune function is unclear. In the study, CiCYP1A gene was cloned from grass carp (Ctenopharyngodon idella). Tissue distribution exhibited an overwhelmingly high basal expression levels in the liver. After GCRV infection, CiCYP1A showed a potent response, indicating CiCYP1A was involved in GCRV-induced immunity. Subcellular localisation showed CiCYP1A was distributed in the cytoplasm. Besides, dual-luciferase activity assays revealed CYP1A was relevant for IFN-I signaling pathway modulation, furthermore, overexpressed CYP1A potently suppressed the mRNA expression of IRF3 and IFN-I but not IRF7. The results provide new sights into exploring immune function of CiCYP1A in teleosts.

Grass carp ATG5 and ATG12 promote autophagy but down-regulate the transcriptional expression levels of IFN-I signaling pathway.

Autophagy is an essential and conserved process that plays an important role in physiological homeostasis, adaptive response to stress and the immune response. Autophagy-related proteins (ATGs) are key components of the autophagic machinery. In the study, grass carp (Ctenopharyngodon idella) autophagy-related gene 5 (ATG5) and 12 (ATG12) were identified. In the gill and intestine, ATG5 and ATG12 were highly expressed, but after grass carp reovirus (GCRV) infection, they were decreased significantly. In Ctenopharyngodon idella kidney (CIK) cells, the sharp variation of ATG5 and ATG12 expression was observed after poly(I:C) infection. Subcellular localisation showed that ATG5 and ATG12 were evenly distributed in the cytoplasm and nucleus. However, the interaction between ATG5 and ATG12 was only found in cytoplasm in both 293T cells and CIK cells. In addition, the overexpression of ATG5 or ATG12 in 293T cells showed enhanced autophagy, and autophagic process was facilitated when ATG5 and ATG12 were simultaneously overexpressed. Dual-luciferase activity assay indicated that both ATG5 and ATG12 remarkably suppressed the promoter activity of IRF3, IRF7, and IFN-I. Further, ATG5 and ATG12 conjugate showed far stronger inhibitory affection on the expression of IFN-I than either ATG5 or ATG12 in response to poly(I:C) or GCRV infection. Taken together, the results demonstrate that grass carp ATG5 and ATG12 play an important role in innate immunity and autophagy.