Gene delivery available in molluscan cells by strong promoter discovered from bivalve-infectious virus.

Understanding gene functions in marine invertebrates has been limited, largely due to the lack of suitable assay systems. Such a system requires investigative methods that are reproducible and can be quantitatively evaluated, such as a cell line, and a strong promoter that can drive high expression of a transgene. In this study, we established primary cell culture from a marine bivalve mollusc, Mizuhopecten yessoensis. Using scallop primary cells, we optimized electroporation conditions for transfection and carried out a luciferase-based promoter activity assay to identify strong promoter sequences that can drive expression of a gene of interest. We evaluated potential promoter sequences from genes of endogenous and exogenous origin and discovered a strong viral promoter derived from a bivalve-infectious virus, ostreid herpesvirus-1 (OsHV-1). This promoter, we termed OsHV-1 promoter, showed 24.7-fold and 16.1-fold higher activity than the cytomegalovirus immediate early (CMV IE) promoter and the endogenous EF1α promoter, the two most commonly used promoters in bivalves so far. Our GFP assays showed that the OsHV-1 promoter is active not only in scallop cells but also in HEK293 cells and zebrafish embryos. The OsHV-1 promoter practically enables functional analysis of marine molluscan genes, which can contribute to unveiling gene-regulatory networks underlying astonishing regeneration, adaptation, reproduction, and aging in marine invertebrates.

Epidemic spreading on spatial higher-order network.

Higher-order interactions exist widely in mobile populations and are extremely important in spreading epidemics, such as influenza. However, research on high-order interaction modeling of mobile crowds and the propagation dynamics above is still insufficient. Therefore, this study attempts to model and simulate higher-order interactions among mobile populations and explore their impact on epidemic transmission. This study simulated the spread of the epidemic in a spatial high-order network based on agent-based model modeling. It explored its propagation dynamics and the impact of spatial characteristics on it. Meanwhile, we construct state-specific rate equations based on the uniform mixing assumption for further analysis. We found that hysteresis loops are an inherent feature of high-order networks in this space under specific scenarios. The evolution curve roughly presents three different states with the initial value change, showing different levels of the endemic balance of low, medium, and high, respectively. Similarly, network snapshots and parameter diagrams also indicate these three types of equilibrium states. Populations in space naturally form components of different sizes and isolations, and higher initial seeds generate higher-order interactions in this spatial network, leading to higher infection densities. This phenomenon emphasizes the impact of high-order interactions and high-order infection rates in propagation. In addition, crowd density and movement speed act as protective and inhibitory factors for epidemic transmission, respectively, and depending on the degree of movement weaken or enhance the effect of hysteresis loops.

Influence of human motion patterns on epidemic spreading dynamics.

Extensive real-data indicate that human motion exhibits novel patterns and has a significant impact on the epidemic spreading process. The research on the influence of human motion patterns on epidemic spreading dynamics still lacks a systematic study in network science. Based on an agent-based model, this paper simulates the spread of the disease in the gathered population by combining the susceptible-infected-susceptible epidemic process with human motion patterns, described by moving speed and gathering preference. Our simulation results show that the emergence of a hysteresis loop is observed in the system when the moving speed is slow, particularly when humans prefer to gather; that is, the epidemic prevalence of the systems depends on the fraction of initial seeds. Regardless of the gathering preference, the hysteresis loop disappears when the population moves fast. In addition, our study demonstrates that there is an optimal moving speed for the gathered population, at which the epidemic prevalence reaches its maximum value.

Identification and functional analysis of transforming growth factor-ß type III receptor (TßR3) from Scylla paramamosain: The first evidence of TßR3 involved in development and innate immunity in invertebrates.

Transforming growth factor-ß type III receptor (TßR3), as a co-receptor of TGF-ß superfamily, plays critical roles in development and growth as well as some disease pathogeneses by presenting ligands to other receptors in vertebrates. However, the identification and functional characterization of TßR3 had not been reported yet in invertebrates. In the present study, TßR3 was first identified and characterized in mud crab Scylla paramamosain. The obtained cDNA length of SpTßR3 was 2, 424 bp with a 1, 854 bp open reading frame, which encoded a putative peptide of 617 amino acids containing a typical transmembrane region and a Zona pellucida (ZP) domain. Real-time PCR results showed that SpTßR3 was predominantly expressed at early embryonic development stage and early postmolt stage, suggesting its participation in development and growth. We report, for the first time in invertebrates, the challenge of both Vibro alginolyticus and Poly (I:C) could alter the expression patterns of SpTßR3. Notably, the expression levels of SpIKK, two NF-κB members (SpRelish and SpDorsal), and five antimicrobial peptide genes (SpCrustin and SpALF1-4) were significantly suppressed when SpTßR3 was interfered in vivo. Secondly, the overexpression of SpTßR3 in vitro could activate NF-κB signaling through the dual-luciferase reporter assays. Furthermore, the bacterial clearance assay after SpTßR3 was silenced in vivo highlighted the potential of SpTßR3 in activating the innate immune responses. These results implied the involvement of SpTßR3 in the innate immune responses by regulating the NF-κB pathway. This study first indicated that TßR3 was present in invertebrate, and it participated in not only the development and growth but also the innate immunity of S. paramamosain. It also provided new insights into the origin or evolution of TGF-ß receptors in crustacean species and even in invertebrates.

Tumor-derived circulating exosomal miR-342-5p and miR-574-5p as promising diagnostic biomarkers for early-stage Lung Adenocarcinoma.

Lung cancer has been the leading cause of cancer morbidity and mortality in recent years. Most lung cancers are often asymptomatic until advanced or metastatic stage. Therefore, looking for the diagnostic biomarker for early-stage lung cancer is quite significant. Circulating exosomal microRNAs (miRNAs) have been reported to be the diagnostic and prognostic markers of various cancers. Here, we obtained circulating exosomal miRNA repertoires of 7 early-stage lung adenocarcinoma patients including pre-operation and post-operation (LA-pre and LA-post) and 7 heathy controls (HCs) by next generation sequence (NGS) and selected miR-342-5p, miR-574-5p and miR-222-3p to validate in ampliative samples by reverse transcription-quantitative PCR (RT-qPCR). Circulating exosomal miR-342-5p, miR-574-5p and miR-222-3p not only significantly elevated in LA patients (n = 56) compared with HCs (n = 40), but also significantly decreased after tumor resection when analyzed 51 paired pre- and post-operation samples. Furthermore, miR-342-5p and miR-574-5p, but not miR-222-3p, had a significantly elevated expression level in carcinoma tissue compared with adjacent non-cancerous tissue (n = 8). The receiver operating characteristic (ROC) curve showed the area under the curve (AUC) of combined miR-342-5p and miR-574-5p was 0.813 (95% CI: 0.7249 to 0.9009) with sensitivity and specificity of 80.0% and 73.2% respectively. In summary, circulating exosomal miR-342-5p and miR-574-5p have potential to serve as novel diagnostic biomarkers for early-stage LA.

Transforming growth factor-ß-activating kinase 1 and its binding protein 1 participate in the innate immune responses via modulating the IMDNFκB signaling in mud crab (Scylla paramamosain).

Transforming growth factor-ß-activating kinase 1 (TAK1) is essential for diverse important biological functions, such as innate immunity, development and cell survival. In the present study, the homologs of TAK1 and TAK1-binding protein 1 (TAB1) were identified and characterized from mud crab Scylla paramamosain for the first time. The full-length cDNAs of SpTAK1 and SpTAB1 were 2, 226 bp and 2, 433 bp with 1, 782 bp and 1, 533 bp open reading frame (ORF), respectively. The deduced SpTAK1 protein contained a conserved S_TKc (Serine/threonine protein kinases, catalytic) domain, and the putative SpTAB1 protein possessed a typical PP2Cc (Serine/threonine phosphatases, family 2C, catalytic) domain and a potential TAK1 docking motif. Real-time PCR analysis showed that SpTAK1 and SpTAB1 were highly expressed at early development stages, suggesting their participation in crab's development process. Moreover, the expression levels of SpTAK1 and SpTAB1 in hepatopancreas were positively stimulated after challenge with Vibro alginolyticus and Poly (I:C), implying the involvement of SpTAK1 and SpTAB1 in innate immune responses against both bacterial and viral infections. When SpTAK1 or SpTAB1 were silenced in vivo, the expression levels of two IMDNFκB signaling components (SpIKKß and SpRelish) and six antimicrobial peptide (AMP) genes (SpALF1-5 and SpCrustin) were significantly reduced, and the bacteria clearance capacity of crabs was also markedly impaired in SpTAK1 or SpTAB1 silenced crabs. Additionally, overexpression of SpTAK1 and SpTAB1 in HEK293T cells could markedly activate the mammalian NF-κB signaling. Collectively, our results suggested that TAK1 and TAB1 regulated crab's innate immunity via modulating the IMDNFκB signaling. These findings may provide new insights into the TAK1/TAB1-mediated signaling cascades in crustaceans and pave the way for a better understanding of crustacean innate immune system.

Effects of both cold and heat stress on the liver of the giant spiny frog (Quasipaa spinosa): stress response and histological changes.

Ambient temperature-associated stress can affect normal physiological functions in ectotherms. To assess the effects of cold or heat stress on amphibians, giant spiny frogs (Quasipaa spinosa) were acclimated at 22°C followed by exposure to 5°C or 30°C for 0, 3, 6, 12, 24 and 48 h, respectively. Histological alterations, apoptotic index, generation of mitochondrial reactive oxygen species (ROS), antioxidant activity indices and stress-response gene expression in frog livers were subsequently determined. Results showed that many fat droplets appeared after 12 h of heat stress and the percentage of melanomacrophage centres significantly changed after 48 h at both stress conditions. Furthermore, the mitochondrial ROS levels were elevated in a time-dependent manner up to 6 h and 12 h in the cold and heat stress groups, respectively. The activities of superoxide dismutase, glutathione peroxidase and catalase were successively increased with increasing periods of cold or heat exposure, and their gene expression levels showed similar changes in both stress conditions. Most tested heat shock protein (HSP) genes were sensitive to temperature exposure, and the expression profiles of most apoptosis-related genes was significantly upregulated at 3 and 48 h under cold and heat stress, respectively. Apoptotic index at 48 h under cold stress was significantly higher than that under heat stress. Notably, lipid droplets, HSP30, HSP70 and HSP110 might be suitable bioindicators of heat stress. The results of these alterations at physiological, biochemical and molecular levels might contribute to a better understanding of the stress response of Q. spinosa, and perhaps amphibians more generally, under thermal stress.

Identification and functional analysis of immune deficiency (IMD) from Scylla paramamosain: The first evidence of IMD signaling pathway involved in immune defense against bacterial infection in crab species.

Immune deficiency (IMD) pathway, one of the most essential pattern recognition receptor signaling pathways, plays vital roles in innate immune responses to eliminate pathogen infection in invertebrates. In the present study, an immune deficiency (IMD) gene and two NF-κB family members, Relish and Dorsal, were identified and characterized in mud crab Scylla paramamosain for the first time. The deduced SpIMD, SpRelish and SpDorsal protein contained conserved death domain and classical NF-κB domains, respectively. Phylogenetic analysis suggested that SpIMD was classified into the invertebrate IMD branch, and SpRelish could be classified into the type I NF-κB class while SpDorsal could be grouped into the type II NF-κB class. Tissue distribution results showed these three genes were ubiquitously expressed in all tested tissues. The expression patterns of IMD signaling pathway and NF-κB genes, including SpIMD, SpIKKß, SpIKKε, SpRelish and SpDorsal, were distinct when crabs were stimulated with Vibro alginolyticus, indicating that they might be involved in responding to bacterial infection. When SpIMD was silenced by in vivo RNA interference assay, the expression levels of IMD pathway and antimicrobial peptides (AMPs) genes, including SpIKKß, SpRelish, SpALF1-6 and SpCrustin, were significantly down-regulated (p < 0.05). Correspondingly, the bacteria clearance ability of hemolymph was extremely impaired in IMD silenced crabs. Overall, the IMD played vital roles in innate immune response by regulating the expressions of its down-stream signaling genes and AMPs in S. paramamosain. These findings might pave the way for a better understanding of innate immune system and establish a fundamental network for the IMD signaling pathway in crustaceans.

Hemocytes of the mud crab Scylla paramamosain: Cytometric, morphological characterization and involvement in immune responses.

Hemocytes play essential roles in the innate immune system of crustaceans. Characterization of hemocytes from estuary mud crab Scylla paramamosain was performed by flow cytometry and morphological studies such as cytochemical staining and electron microscopy. The hemocyte subsets were further separated using a modified Percoll density gradient centrifugation method. Based on the morphological characteristics of the cells, three distinct categories of hemocytes were identified: granulocytes with abundant large granularity representing 5.27 ± 0.42%, semigranulocytes with small or less granularity representing 76.03 ± 3.34%, and hyalinocytes (18.70 ± 3.92%) which were almost no granularity. The total hemocyte cell count and the percentage of hemocyte subsets varied after pathogen infection, including Vibrio alginolyticus and the viral double-stranded RNA analog Poly (I:C). The phagocytic process is of fundamental importance for crustaceans' cellular immune response as well as development and survival. The results of the in vitro phagocytosis assays analyzed by flow cytometry demonstrated that granulocytes and semigranulocytes had significantly higher phagocytic ability than hyalinocytes. A primary culture system, L-15 medium supplemented with 5-10% fetal bovine serum, was developed to further investigate the immune function of hemocytes. Furthermore, adenovirus can be utilized to effectively transfer GFP gene into hemocytes. Overall, three hemocyte sub-populations of S. paramamosain were successfully discriminated, moreover, their response to pathogen infections, phagocytic activity and adenovirus mediated transfection were also investigated for the first time. This study may contribute to a better understanding of the innate immune system of estuary crabs.

Effects of heat stress on the liver of the Chinese giant salamander Andrias davidianus: Histopathological changes and expression characterization of Nrf2-mediated antioxidant pathway genes.

Nuclear factor E2-related factor 2 (Nrf2) is a crucial transcription factor that regulates the basal and inducible expression of many antioxidant-relevant genes, and the Nrf2-mediated antioxidant pathway has been regarded as a critical switch in the initiation of cellular defence systems against oxidative damages. In this study, Nrf2 was first identified and characterized in the Chinese giant salamander (Andrias davidianus). A. davidianus was exposed to a high ambient temperature of 30 °C for various periods of time (0, 3, 6, 12, 24, 48 and 72 h). We investigated the effects of heat stress on alterations of the hepatic malondialdehyde (MDA) concentration, the activities of lactic acid dehydrogenase (LDH), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), the histology of the liver, and the mRNA expression patterns of 11 genes involved in the Nrf2-mediated antioxidant pathway in A. davidianus. The results showed that both the hepatic LDH activity and MDA content significantly increased after heat exposure, indicating that heat stress could induce cell injury and oxidative damage. Histological analysis of the liver showed that heat stress caused hepatocyte abnormalities, fat accumulation and ultrastructural alterations of the hepatocytes, endoplasmic reticulum and nuclei. The expression patterns of genes involved in the Nrf2-mediated antioxidant pathway in the liver were distinct when A. davidianus was exposed to heat stress. To the best of our knowledge, this study is the first on the characterization of Nrf2 in A. davidianus and even in amphibians. The results indicated that heat stress could induce oxidative damage, and the Nrf2 antioxidant pathway might play a critical role in the resistance against heat stress in A. davidianus. These findings will deepen and enrich the current knowledge on the evolutionary conserved antioxidant roles and mechanisms of Nrf2 in A. davidianus, or even in amphibians, in the antioxidant defence against heat stress.

Identification and characterization of atypical 2-cysteine peroxiredoxins from mud crab Scylla paramamosain: The first evidence of two peroxiredoxin 5 genes in non-primate species and their involvement in immune defense against pathogen infection.

Peroxiredoxin 5 (Prx5) belongs to a novel family of evolutionarily conserved antioxidant proteins that protect cells against various oxidative stresses. Generally, no more than one Prx5 transcript had been reported in non-primate species. In this study, two Prx5 genes (coined as SpPrx5-1 and SpPrx5-2) were firstly isolated from the mud crab, Scylla paramamosain, through RT-PCR and RACE methods. The open reading frame of SpPrx5-1 and SpPrx5-2 were 561 bp and 429 bp in length, encoding 186 and 142 amino acids polypeptide, respectively. Both the conserved signatures of peroxiredoxin catalytic center and Prx5-specific domain were identified in SpPrx5-1 and SpPrx5-2. Phylogenetic analysis indicated that both SpPrx5 clustered together with other animal Prx proteins and were classified into Prx5 subfamily. Tissue-specific expression analysis revealed that both SpPrx5-1 and SpPrx5-2 were ubiquitously expressed, highest in hepatopancreas, and showed remarkably similar transcription patterns. Quantitative RT-PCR analysis exhibited that both SpPrx5 genes changed dramatically in hepatopancreas, although showing different expression profiles, after virus-analog poly (I:C) or Vibrio alginolyticus challenge. The expression levels of both SpPrx5s were significantly enhanced in hepatopancreas after poly (I:C) stimulation, while SpPrx5-2 exhibited a more prompt response than SpPrx5-1. Nevertheless, the expression levels of both SpPrx5s were significantly reduced in hepatopancreas after Vibrio alginolyticus challenge in which SpPrx5-1 showed a more prompt response than SpPrx5-2. These results suggested the involvement of SpPrx5s in responses against viral and bacterial infections and further highlighted their functional importance in the immune system of Scylla paramamosain.

Identification and characterization of pro-interleukin-16 from mud crab Scylla paramamosain: The first evidence of proinflammatory cytokine in crab species.

IL-16 is a pro-inflammatory cytokine originally designated as a lymphocyte chemoattractant factor. In mammal and avian, it has been characterized as an essential regulator of various cellular processes including cell recruitment and activation against pathogen invasion. So far, neither of the full-length of IL-16 homologue nor the response mechanism against pathogen was reported in crab species. In the present study, the pro-IL-16 homologue was firstly cloned and characterized from mud crab Scylla paramamosain. The full-length Sp-pro-IL-16 consisted of 4107 bp with an opening reading frame encoding 1369 amino acids. Multiple alignment analysis showed the putative amino acid sequence of Sp-pro-IL-16 had about 73.86% identity with Litopenaeus vannamei pro-IL-16. Additionally, two conserved PDZ domains and protein binding sites were found in Sp-pro-IL-16 and showed high similarities about 94.19% and 51.14% with their Litopenaeus vannamei and Mus musculus counterparts. RT-PCR analysis indicated that Sp-pro-IL-16 transcripts were constitutively expressed in all tissues examined with an extreme high level in hepatopancreas. Moreover, Sp-pro-IL-16 transcripts in hepatopancreas were significantly up-regulated 15-fold at 72 h after Vibrio alginolyticus challenge and 3.5-fold at 12 h after virus-analog Poly (I:C) challenge. The Western blot analysis revealed that Sp-pro-IL-16 can be cleaved to its bioactive form, an approximately 35 kDa mature IL-16, and the protein levels of both pro-IL-16 and mature IL-16 increased after Vibrio alginolyticus challenge. It is the first experimental identification of pro-inflammatory cytokine IL-16 in arthropods. This study could shed new light on further understanding of the response mechanism of pro-inflammatory cytokine IL-16 in Scylla paramamosain against pathogens. Meanwhile, it brought new insight into the origin and evolution of IL-16 in crab species.

Expression and functional analyses for estrogen receptor and estrogen related receptor of Yesso scallop, Patinopecten yessoensis.

Estrogen receptors (ERs) were known as estrogen-activated transcription factors and function as major reproduction regulators in vertebrates. The presence of er genes had been reported in Molluscan cephalopods and gastropods. However, they were considered as constitutive activators with unknown biological functions since reporter assays for these ERs did not show a specific response to estrogens. In this study, we tried characterization of ER orthologues from the Yesso scallop, Patinopecten yessoensis, in which estrogens had been proven to be produced in the gonads and involved in the spermatogenesis and vitellogenesis. Identified ER and estrogen related receptor (ERR) of Yesso scallops, designated as py-ER and py-ERR, conserved specific domain structures for a nuclear receptor. Their DNA binding domains showed high similarities to those of vertebrate ER orthologues, while ligand binding domains had low similarities with them. Both the py-er and py-err expression levels decreased in the ovary at the mature stage while py-vitellogenin expression increased in the ovary by quantitative real-time RT-PCR. Also, the py-er and py-err showed higher expressions in the testis than ovary during the developing and mature period, suggesting both genes might function in the spermatogenesis and testis development. The py-ER showed binding affinities to vertebrate estradiol-17ß (E2). However, the intensity was weaker than the vertebrate ER, indicating scallops might exist endogenous estrogens with a different structure. On the other hand, the binding property of py-ERR to E2 was not confirmed in this assay, speculating that py-ERR was a constitutive activator as other vertebrate ERRs. Further, the py-er was localized in the spermatogonia in the testis and in the auxiliary cells in the ovary by in situ hybridization, indicating its potential roles in promoting spermatogenesis and vitellogenesis. Taken together, the present study demonstrated that py-ER was an authentic E2 receptor in the Yesso scallop and might have functions for the spermatogonia proliferation and vitellogenesis, while py-ERR was involved in the reproduction by undiscovered manners.

Proton Magnetic Resonance Spectroscopy for the Early Diagnosis of Parkinson Disease in the Substantia Nigra and Globus Pallidus: A Meta-Analysis With Trial Sequential Analysis.

This study aimed to investigate the metabolic changes in globus pallidus (GP) and substantia nigra (SN) during the early stage of Parkinson disease (PD) using magnetic resonance spectroscopy (MRS). PubMed, Embase, Web of Science, and Chinese National Knowledge Infrastructure were searched till November 2018. Eligible trials comparing early metabolic changes in GP and SN in patients with PD vs. controls were included. The mean differences with 95% confidence intervals were estimated with either fixed- or random-effects models using Review Manager 5.3 software. Trial sequential analysis was performed using TSA 0.9.5.10 beta software. Finally, 16 studies were selected from the search. Overall, the N-acetyl aspartate-to-creatine ratio showed a significant difference between patients with early-stage PD and healthy controls. The overall heterogeneity was P < 0.00001, I 2 = 94% in GP and P = 0.0002, I 2 = 74% in SN. The results revealed that MRS could be a more sensitive imaging biomarker in the diagnosis of early-stage PD. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=125731, registration number: CRD42019125731.

The long noncoding RNA HOTAIRM1 controlled by AML1 enhances glucocorticoid resistance by activating RHOA/ROCK1 pathway through suppressing ARHGAP18.

Acquired resistance to glucocorticoids (GCs) is an obstacle to the effective treatment of leukemia, but the molecular mechanisms of steroid insensitivity have not been fully elucidated. In this study, we established an acquired GC-resistant leukemia cell model and found a long noncoding RNA, HOTAIRM1, was overexpressed in the resistant cells by transcriptional profiling, and was higher expressed in patients with poor prognosis. The whole-genome-binding sites of HOTAIRM1 were determined by ChIRP-seq (chromatin isolation by RNA purification combined with sequencing) analysis. Further study determined that HOTAIRM1 bound to the transcriptional inhibitory region of ARHGAP18 and repressed the expression of ARHGAP18, which led to the increase of RHOA/ROCK1 signaling pathway and promoted GC resistance through antiapoptosis of leukemia cells. The inhibition of ROCK1 in GC-resistant cells could restore GCs responsiveness. In addition, HOTAIRM1 could also act as a protein sequester to prevent transcription factor AML1(acute myeloid leukemia 1) from binding to the regulatory region of ARHGAP18 by interacting with AML1. At last, we also proved AML1 could directly activate the expression of HOTAIRM1 through binding to the promoter of HOTAIRM1, which enriched the knowledge on the regulation of lncRNAs. This study revealed epigenetic causes of glucocorticoid resistance from the perspective of lncRNA, and laid a foundation for the optimization of glucocorticoid-based leukemia treatment strategy in clinic.

Functional differences in the products of two TRAF3 genes in antiviral responses in the Chinese giant salamander, Andrias davidianus.

Tumour necrosis factor receptor associated factor 3 (TRAF3) is a crucial transducing protein for linking upstream receptor signals and downstream antiviral signalling pathways. Previous studies mostly clarified the functions of TRAF3 in mammals, birds and fish, but little is known about the characterization and function of TRAF3 in amphibians. In this study, the molecular and functional identification of two TRAF3 genes, AdTRAF3A and AdTRAF3B, were investigated in the Chinese giant salamander Andrias davidianus. The complete open reading frames (ORFs) of AdTRAF3A and AdTRAF3B were 1698 bp and 1743 bp in length, encoding 565 and 580 amino acids, respectively. Both AdTRAF3A and AdTRAF3B deduced proteins contained a RING finger, two TRAF-type zinc fingers, a coiled-coil and a MATH domain. Phylogenetic analysis showed that the AdTRAF3 protein clustered together with other known TRAF3 proteins. Gene expression analysis showed that AdTRAF3s were broadly distributed in all examined tissues with similar distribution patterns. AdTRAF3s in the blood or spleen positively responded to Giant salamander iridovirus (GSIV) and poly (I:C) induction but exhibited distinct response patterns. Silencing AdTRAF3A/B remarkably suppressed the expression of IFN signalling pathway-related genes when leukocytes were treated with DNA virus and the viral RNA analogue. Moreover, overexpression of AdTRAF3A may induce the activation of the IFN-ß promoter, and the zinc finger, coiled coil and MATH domains of AdTRAF3A were essential for IFN-ß promoter activation. However, the overexpression of AdTRAF3B significantly suppressed IFN-ß promoter activity, and its inhibitory effect was enhanced when the RING finger or MATH domain was deleted. Furthermore, AdTRAF3A rather than AdTRAF3B significantly induced NF-κB activation, implying that AdTRAF3A may function as an enhancer in both the IFN and NF-κB signalling pathways. Taken together, our results suggest that the two TRAF3 genes play different crucial regulatory roles in innate antiviral immunity in Chinese giant salamanders.

A Study on Microstructure, Residual Stresses and Stress Corrosion Cracking of Repair Welding on 304 Stainless Steel: Part I-Effects of Heat Input.

In this paper, the effect of repair welding heat input on microstructure, residual stresses, and stress corrosion cracking (SCC) sensitivity were investigated by simulation and experiment. The results show that heat input influences the microstructure, residual stresses, and SCC behavior. With the increase of heat input, both the δ-ferrite in weld and the average grain width decrease slightly, while the austenite grain size in the heat affected zone (HAZ) is slightly increased. The predicted repair welding residual stresses by simulation have good agreement with that by X-ray diffraction (XRD). The transverse residual stresses in the weld and HAZ are gradually decreased as the increases of heat input. The higher heat input can enhance the tensile strength and elongation of repaired joint. When the heat input was increased by 33%, the SCC sensitivity index was decreased by more than 60%. The macroscopic cracks are easily generated in HAZ for the smaller heat input, leading to the smaller tensile strength and elongation. The larger heat input is recommended in the repair welding in 304 stainless steel.

Functional differences of three CXCL10 homologues in the giant spiny frog Quasipaa spinosa.

Chemokines are a superfamily of structurally related chemotactic cytokines exerting significant roles in acting as a bridge between the innate and adaptive immune responses. In this study, we identified three CXC motif chemokine 10 (CXCL10) homologues (QsCXCL10-1, QsCXCL10-2 and QsCXCL10-3) from giant spiny frog Quasipaa spinosa. All three deduced QsCXCL10 proteins contained four conserved cysteine residues as found in other known CXC chemokines. Phylogenetic analysis showed that QsCXCL10-1, 2, 3 and other CXCL10s in amphibian were grouped together to form a separate clade. These three QsCXCL10s were highly expressed in spleen and blood. Upon infection with Staphylococcus aureus or Aeromonas hydrophila, the expressions of QsCXCL10s were markedly increased in spleen and blood during biotic stresses. Meanwhile, the QsCXCL10s transcription in liver could also be up-regulated under abiotic stresses such as cold and heat stresses. The recombinant proteins of frog CXCL10 homologues were produced and purified in E. coli and possessed similar but differential bioactivities. Both rCXCL10-1 and rCXCL10-2 had strong effects on the up-regulation of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-8) in vivo, whereas rCXCL10-3 induced a weak expression of these cytokines. Moreover, the rCXCL10-1 and rCXCL10-2 could strongly promote splenocyte proliferation and induce lymphocytes migration, while rCXCL10-3 had limited effects on these biological processes. All three frog chemokines triggered their functional activities by engaging CXC motif chemokine receptor 3 (CXCR3). Taken together, these results revealed that the three QsCXCL10s had similar but differential functional activities in mediating immune responses and host defenses, which might contribute to a better understanding of the functional evolution of CXCL10 in vertebrates.

Identification and functional analysis of two interferon regulatory factor 3 genes and their involvement in antiviral immune responses in the Chinese giant salamander Andrias davidianus.

Interferon regulatory factor 3 (IRF3), a crucial member of interferon regulatory factor (IRF) family, plays an important role in innate immunity in vertebrates. However, there are no reports on the characterization and especially their respective functional analysis of two IRF3 genes in some species. In this study, two IRF3 genes as well as their roles in the immune response were identified and investigated in Chinese giant salamander, Andrias davidianus. The complete open reading frames of AdIRF3A and AdIRF3B were 1, 113 bp and 1, 380 bp in length, encoding 370 and 459 amino acids, respectively. Both AdIRF3A and AdIRF3B protein contain an IRF and an IRF3 domain. Phylogenetic analysis indicated that AdIRF3s clustered together with other IRF3 proteins. Tissue distribution analysis showed that AdIRF3s were expressed in all tissues tested, with highest expression levels in blood. Both AdIRF3s actively responded to Chinese giant salamander iridovirus (GSIV) and poly (I:C) challenge in A. davidianus. AdIRF3A/B silencing significantly suppressed the DNA virus and viral RNA analog-induced expression of IFN-inducible genes. Luciferase reporter assay further confirmed the regulatory role of AdIRF3s in IFN signaling. These results provide new insights into the origin or evolution of IRF3 in amphibians and even in vertebrates.

The first evidence of four transcripts from two Interleukin 18 genes in animal and their involvement in immune responses in the largest amphibian Andrias davidianus.

Interleukin 18 (IL-18), a member of IL-1 cytokine superfamily, is an important proinflammatory cytokine with multiple functions in both innate immunity and acquired immunity. However, the characteristics and functional roles of IL-18 remain largely unknown in amphibians, which were classed as major group of vertebrates. In the present study, two IL-18 genes (AdIL-18A and AdIL-18B) and four transcripts (AdIL-18A1, AdIL-18A2, AdIL-18B1 and AdIL-18B2) were firstly identified and characterized from Chinese giant salamander (Andrias davidianus). To the best of our knowledge, this is the first report on the presence of more than one gene copy or two transcripts of IL-18 in one species. The complete open reading frames of AdIL-18A1, AdIL-18A2, AdIL-18B1 and AdIL-18B2 were 588 bp, 603 bp, 591 bp and 606 bp, respectively. The putative AdIL-18 proteins possessed the typical IL-1 domains and phylogenetic analysis indicated that AdIL-18s grouped together with other vertebrate IL-18 proteins. The expression profiles of AdIL-18s were investigated under the challenges of Aeromonas hydrophila, Staphylococcus ureae and Poly (I:C) respectively, and the results suggested that AdIL-18s were involved in the immune responses against both bacterial and viral infections. Moreover, the expression levels of two NF-κBs (P100 and P105) and four proinflammatory cytokines (IL-1ß, IL-6, TNF-α and IFN-γ) were inhibited in AdIL-18A1/A2-silenced cells when treated with bacteria and viral RNA analog. Additionally, the transcription levels of these immune-related cytokine genes were markedly induced when the lymphocytes were treated with recombinant AdIL-18A1 or AdIL-18A2 proteins, implying the involvement of AdIL-18s in triggering NF-κB signaling and proinflammatory responses. These results might provide new insights into the origin or evolution of IL-18 in amphibians and even in vertebrates.