Characterization of type II IFNs and their receptors in a cyprinid fish, the blunt snout bream Megalobrama amblycephala.

Type II interferons (IFNs) are a key class of molecules regulating innate and adaptive immunity in vertebrates. In the present study, two members of the type II IFNs, IFN-γ and IFNγ-rel, were identified in the blunt snout bream (Megalobrama amblycephala). The open reading frame (ORF) of IFN-γ and IFNγ-rel was found to have 564 bp and 492 bp, encoding 187 and 163 amino acids, with the first 26 and 24 amino acids being the signal peptide, respectively. IFN-γ and IFNγ-rel genes showed a high degree of similarity to their zebrafish homologues, being 76.9 % and 58.9 %, respectively. In the phylogenetic tree, IFN-γ and IFNγ-rel were clustered with homologous genes in cyprinids. In blunt snout bream, IFN-γ and IFNγ-rel were constitutively expressed in trunk kidney, head kidney, spleen, liver, heart, muscle, gill, intestine and brain and were significantly up-regulated by poly (I:C) induction in head kidney, spleen, liver, gill and intestine. Using recombinant proteins of IFN-γ and IFNγ-rel, the surface plasmon resonance (SPR) results showed that IFN-γ was bound to CRFB6, CRFB13 and CRFB17, but mainly to CRFB6 and CRFB13, whereas IFN-γrel bound mainly to CRFB17 and had no affinity with CRFB6. These results contribute to a better understanding on type II IFNs and their receptor usage in teleost fish.

A CT-based radiomics nomogram for distinguishing between malignant and benign Bosniak IIF masses: a two-centre study.

AIM: To establish and assess a computed tomography (CT)-based radiomics nomogram for identifying malignant and benign Bosniak IIF masses. MATERIALS AND METHODS: In total, 150 patients with Bosniak IIF masses were separated into a training set (n=106) and a test set (n=44) in a ratio of 7:3. A radiomics signature was calculated based on extracted features from the three phases of CT images. A clinical model was constructed based on clinical characteristics and CT features, and a nomogram incorporating the radiomics signature and independent clinical variables was established. The calibration ability, discrimination accuracy, and clinical value of the nomogram model were assessed. RESULTS: Twelve features derived from CT images were applied to establish the radiomics signature. The performance levels of three machine-learning models were improved by adding the synthetic minority oversampling technique algorithm. The optimised machine learning model was a combination of the minimum redundancy maximum relevance-least absolute shrinkage and selection operator feature screening method + logistic regression classifier + synthetic minority oversampling technique algorithm, which demonstrated excellent identification ability on the test set (area under the curve [AUC], 0.970; 95% confidence interval [CI], 0.940-1.000). The nomogram model displayed outstanding discrimination ability on the test set (AUC, 0.972; 95% CI, 0.942-1.000). CONCLUSIONS: The CT-based radiomics nomogram was useful for discriminating between malignant and benign Bosniak IIF masses, which improved the precision of preoperative diagnosis.

Dung treated by high-temperature composting is an optimal bedding material for suckling calves according to analyses of microbial composition, growth performance, health status, and behavior.

Bedding materials are important for suckling buffalo calves. Treated dung has been used as a bedding material for dairy cows but the lack of an appropriate safety assessment limits its application. In this study, we evaluated the feasibility of treated dung (TD) as a bedding material for suckling calves by comparing TD with rice husk (RH) and rice straw (RS) bedding materials. The TD was prepared through high-temperature composting by Bacillus subtilis. Thirty-three newborn suckling buffalo calves (Bubalus bubalis, 40.06 ± 5.79 kg) were randomly divided into 3 bedding material groups (TD, RH, and RS) and bedded with 1 of the 3 bedding materials for 60 d. We compared cost, moisture content, bacterial counts, and microbial composition of the 3 bedding materials, and investigated growth performance, health status, behavior, rumen fermentation, and blood parameters of bedded calves. The results showed that TD contained the fewest gram-negative bacteria and coliforms on d 1 and 30 and the lowest relative abundance of Staphylococcus throughout the experiment. The RH and TD bedding materials had the lowest cost. Calves in the TD and RS groups showed a higher dry matter intake, and final body weight and average daily gain in the TD and RS groups tended to be higher than in the RH group. Calves in the TD and RS groups had a lower disease incidence (diarrhea and fever), fewer antibiotic treatments, and lower fecal score than calves in the RH group. Higher contents of IgG, IgA, and IgM were observed in calves of the TD and RS groups than in calves of the RH group on d 10, indicating higher immune ability in TD and RS groups. Furthermore, TD bedding increased the butyric acid content in the calf's rumen, whereas RS bedding increased the acetate content, which might be attributed to the longer time and higher frequency of eating bedding material in the RS group. Considering all of the above indicators, we concluded that TD is the optimal bedding material for calves based on economics, bacterial count, microbial diversity, growth performance, and health status. Our findings provide a valuable reference for bedding material choice and calf farming.

Four type I IFNs, IFNa1, IFNa2, IFNb, IFNc, and their receptor usage in an osteoglossomorph fish, the Asian arowana, Scleropages formosus.

In fish, type I IFNs are classified into three groups, i.e. Group I, Group II and Group III, which are further divided into seven subgroups according to the number of conservative cysteines, phylogenetic relationship, and probably their receptor complexes. In the present study, four type I IFNs and four cytokine receptor family B members (CRFBs) were identified in the Asian arowana, Scleropages formosus, an ancient species in the Osteoglossomorpha with commercial and conservation values. According to multiple sequence alignment and phylogenetic relationship, the four type I IFNs are named as IFNa1, IFNa2, IFNb and IFNc, with the former two belonging to Group I, and the latter two to Group II. The four receptors are named as CRFB1, CRFB2, CRFB5a and CRFB5b. The IFNs and their possible receptor genes are widely expressed in examined organs/tissues, and are induced following the stimulation of polyinosinic polycytidylic acid (polyI:C) in vivo. It was found that IFNa1, IFNa2, IFNb and IFNc use preferentially the receptor complexes, CRFB1 and CRFB5b, CRFB1 and CRFB5b, CRFB2 and CRFB5a, and CRFB2 and CRFB5b, respectively, indicating the evolutionary diversification in the interaction of type I IFNs and their receptors in this ancient fish species, S. formosus.

Radiomics-based machine-learning method for prediction of distant metastasis from soft-tissue sarcomas.

AIM: To construct and validate a radiomics-based machine-learning method for preoperative prediction of distant metastasis (DM) from soft-tissue sarcoma. MATERIALS AND METHODS: Seventy-seven soft-tissue sarcomas were divided into a training set (n=54) and a validation set (n=23). The performance of three feature selection methods (ReliefF, least absolute shrinkage and selection operator [LASSO], and regularised discriminative feature selection for unsupervised learning [UDFS]) and four classifiers, random forest (RF), logistic regression (LOG), K nearest neighbour (KNN), and support vector machines (SVMs), were compared for predicting the likelihood of DM. To counter the imbalance in the frequencies of DM, each machine-learning method was trained first without subsampling, then with the synthetic minority oversampling technique (SMOTE). The performance of the radiomics model was assessed using area under the receiver-operating characteristic curve (AUC) and accuracy (ACC) values. RESULTS: The performance of the LASSO and SVM algorithm combination used with SMOTE was superior to that of the algorithm combination alone. The combination of SMOTE with feature screening by LASSO and SVM classifiers had an AUC of 0.9020 and ACC of 91.30% in the validation dataset. CONCLUSION: A machine-learning model based on radiomics was favourable for predicting the likelihood of DM from soft-tissue sarcoma. This will help decide treatment strategies.

Mental distress and its associations with behavioral outcomes during the COVID-19 pandemic: a national survey of Chinese adults.

OBJECTIVES: This study aimed to evaluate associations between mental distress and COVID-19-related changes in behavioral outcomes and potential modifiers (age, gender, educational attainment) of such associations. STUDY DESIGN: This was a cross-sectional study. METHODS: An online survey using anonymous network sampling was conducted in China from April to May 2020 using a 74-item questionnaire. A national sample of 10,545 adults in 31 provinces provided data on sociodemographic characteristics, COVID-19-related mental distress, and changes in behavioral outcomes. Structural equation models were used for data analyses. RESULTS: After adjusting for covariates, greater mental distress was associated with increased smoking (odds ratio [OR] = 1.42, 95% confidence interval [CI]: 1.20-1.68 and OR = 1.54, 95% CI: 1.31-1.82 per one standard deviation [SD] increase in mental distress) and alcohol consumption (OR = 1.67, 95% CI: 1.45-1.92 and OR = 1.47, 95% CI: 1.24-1.75 per one SD increase in mental distress) among current smokers and drinkers and with both increased and decreased physical activity (ORs ranged from 1.32 to 1.56). Underweight adults were more likely to lose body weight (≥1 kg; OR = 1.63, 95% CI: 1.30-2.04), whereas overweight adults were more likely to gain weight (OR = 1.61, 95% CI: 1.46-1.78) by the same amount. Association between mental distress and change in physical activity was stronger in adults aged ≥40 years (ORs ranged from 1.43 to 2.05) and those with high education (ORs ranged from 1.43 to 1.77). Mental distress was associated with increased smoking in males (OR = 1.60, 95% CI: 1.37-1.87) but not females (OR = 1.11, 95% CI: 0.82-1.51). CONCLUSIONS: Greater mental distress was associated with some positive and negative changes in behavioral outcomes during the pandemic. These findings inform the design of tailored public health interventions aimed to mitigate long-term negative consequences of mental distress on outcomes.

Expression and antibacterial analysis of galectin-8 and -9 genes in mandarin fish, Siniperca chuatsi.

Galectin-8 and galectin-9 belong to tandem repeat-type galectins, and in the present study, these two genes were cloned in mandarin fish Siniperca chuatsi. The open reading frame (ORF) of the mandarin fish galectin-8 and galectin-9 contains 942, and 1008 bp, encoding 313 and 335 amino acids, respectively. As a conserved feature, an N-terminal carbohydrate recognition domain (CRD), and a C-terminal CRD were observed in each of the two galectins in mandarin fish. In healthy fish, galectin-8 and -9 were constitutively expressed in all organs/tissues examined, and their expression can be induced following the stimulation of LPS and poly(I:C). It is obvious that galectin-8 had a higher increase at mRNA level following the stimulation of poly(I:C). It is further demonstrated that mandarin fish galectin-8 inhibited the growth of Flavobacterium columnare and Streptococcus agalactiae, and in addition to the two species of bacteria, galectin-9 inhibited also the growth of Edwardsiella piscicida, which provides the basis for further understanding their antibacterial role in immune response of mandarin fish.

Identification of a novel splice variant isoform of interferon regulatory factor 10, IRF10, in orange spotted grouper Epinephelus coioides.

Interferon regulatory factors (IRFs) are a family of transcription factors essential to the control of antiviral immune response, cell growth, differentiation and apoptosis. IRF10 was originally found in chicken, which was induced by the v-Rel oncoprotein in lymphoid cell lines and involved in the upregulation of major histocompatibility complex (MHC) class I and guanylate-binding protein. In fish, IRF10 plays negative roles in regulation of the interferon (IFN) response. Here, we identified a splice variant of IRF10, named as EcIRF10-SF in orange spotted grouper, which shares the first three exons with the long form (EcIRF10-LF) and retains part of intron 3, creating a premature termination codon. Furthermore, we observed that the EcIRF10-SF exhibits similar expression pattern compared to its native counterparts. Functional studies demonstrate that the two EcIRF10 isoforms repress DrIFNϕ1 and DrIFNϕ3 promoter activity and negatively regulate fish antiviral gene expression. Subcellular localization analysis shows that the amino acids from 57 to 86 within DBD are required for IRF10 nuclear import. Overall, our description of transcript diversification of IRF10 in the grouper provides a coherent framework to further dissect its roles in immune response.

Identification of type I IFNs and their receptors in a cyprinid fish, the topmouth culter Culter alburnus.

In fish, type I IFNs are classified into three groups, i.e. group one, group two and group three, and further separated into seven subgroups based on the number of conserved cysteines and phylogenetic relationships. In the present study, four type I IFNs, named as IFNϕ1, IFNϕ2, IFNϕ3, IFNϕ4, as reported in zebrafish, were identified in a cyprinid, the topmouth culter, Culter alburnus, a species introduced recently into China's aquaculture. These IFNs may be classified as IFNa, IFNc, IFNc and IFNd in a recent nomenclature, with IFNa and IFNd having two cysteines in group one, and IFNc four cysteines in group two. These IFNs, together with their possible receptors, IFNϕ1, IFNϕ2, IFNϕ3, IFNϕ4, and CRFB1, CRFB2 and CRFB5 have an open reading frame (ORF) of 540, 552, 567, 516 bp, and 1572, 1392, 1125 bp, respectively. These IFNs have high amino acid sequence identities, being 91.1-93.6% and 66.9-77.3%, with those in grass carp and zebrafish, respectively, and are expressed constitutively in organs/tissues examined in the fish. The expression of these IFNs can be further induced following poly (I:C) stimulation. However, the possible function of these IFNs and their signalling pathway are of interest for further research.

Multiple b-value diffusion-weighted imaging in differentiating benign from malignant breast lesions: comparison of conventional mono-, bi- and stretched exponential models.

AIM: To prospectively evaluate multiple b-value diffusion-weighted imaging (DWI) in differentiating malignant from benign breast lesions. MATERIALS AND METHODS: The study cohort included 103 patients who underwent 3 T magnetic resonance imaging (MRI). The conventional sequences included T1-weighted dynamic contrast-enhanced, T1-weighted and T2-weighted fat-suppressed sequences, single b-value (b=0, 1000 s/mm2) DWI, and multiple b-values (12 values, from 0 to 3,000 s/mm2) DWI. Pathological diagnosis of breast lesions was based on the latest World Health Organization (WHO) guide on the pathology and immunohistochemistry of the breast. SPSS Statistics V19.0 was used for the statistics analysis. RESULTS: The following parameters were calculated: apparent diffusion coefficient (ADC), tissue diffusivity (D), perfusion fraction (f), pseudo-diffusion coefficient (D∗), distributed diffusion coefficient (DDC), and alpha (α) by the same radiologist twice (interval time of 3 months). There was good inter/intra-observer agreement for each of the parameters. The D, D∗, f, DDC, and α values were significantly different among malignant tumours, benign lesions, and normal breast tissue (p<0.05). CONCLUSION: D, f, DDC, α, and ADC values have good sensitivity and specificity, respectively. In addition, the combined use of D and f or DDC and α has good diagnostic performance. Thus, the applications of the new multi-b DWI variables or combined variables are promising.

Identification of a novel RIG-I isoform and its truncating variant in Japanese eel, Anguilla japonica.

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic viral RNA sensor that triggers the production of type I interferons (IFNs) and proinflammatory cytokines during viral infection. RIG-I gene has been identified previously in Japanese eel, Anguilla japonica. In the present study, we have characterized a novel isoform of RIG-I (designated as AjRIG-Ib) and its truncated variant (AjRIG-Ibv). The AjRIG-Ib encodes 940 amino acids (aa) consisting of two N-terminal caspase activation and recruitment domains (CARDs), a DEX(D/H) box RNA helicase domain, and a C-terminal regulatory domain (CTD). The AjRIG-Ibv encodes a protein of 843 aa, that shares similar structural organization with AjRIG-Ib, but lacking CTD. The gene expression analyses showed that AjRIG-Ib and AjRIG-Ibv were detectable in all tissues/organs examined, and AjRIG-Ib was the predominant form. The mRNA level of AjRIG-Ibv was upregulated rapidly at 8 h after the Poly I:C injection, and the significant increase of AjRIG-Ib was observed at 16 and 24 h post-injection (hpi). Laser confocal microscopy showed that AjRIG-Ib and AjRIG-Ibv were both located in cytoplasm. In addition, the overexpression of AjRIG-Ib or AjRIG-Ibv led to the increased activity of IFN promoter in transient transfection assay. Taken together, our results indicated that AjRIG-Ib and AjRIG-Ibv may play cooperative or somewhat complementary roles in coordinating the antiviral response in fish.

Characterization of MyD88 in Japanese eel, Anguilla japonica.

Myeloid differentiation factor 88 (MyD88) is a key adaptor protein required for the signaling of all Toll-like receptors except TLR3, which results to the interaction of activated TLR complexes via C-terminal TIR domain and the binding of downstream kinase via N-terminal death domain. In this study, the MyD88 gene from the Japanese eel (Anguilla japonica) was identified. The open reading frame of AjMyD88 was 918 bp in length, encoding a protein composed of conserved N-terminal death domain and C-terminal TIR domain, respectively. Multiple alignment revealed highly conserved sites across all examined vertebrate lineages in death and TIR domains. Site-directed mutagenesis and luciferase analysis revealed that the W78A, L91A and L95A mutations in death domain had modest impairment of their ability in activating NF-κB promoter. The expression level of AjMyD88 was investigated by real-time PCR in response to poly I:C stimulation and Edwardsiella tarda infection. Significantly increased MyD88 expression was observed at early phase in all tested tissues/organs in response to E. tarda infection and slight increase was detected in intestine and gill at 16 hpi and in head kidney, spleen and liver at 24 hpi after poly I:C stimulation. Immunofluorescence staining revealed that AjMyD88 is present as condensed forms in the cytoplasm. Taken together, sequence characterization, gene expression and cellular distribution data obtained in this study suggest that AjMyD88, similar to its mammalian ortholog, plays an important role in eel immune response against bacteria.

Contrast-enhanced CT findings of intravenous leiomyomatosis.

AIM: To characterise the contrast-enhanced computed tomography (CT) imaging findings of nine patients with intravenous leiomyomatosis (IVL). MATERIALS AND METHODS: The contrast-enhanced CT imaging findings of nine patients with histopathologically proven IVL were examined. The location, morphology, extension pathway, adhesion, and degree and pattern of enhancement of the tumour were assessed. RESULTS: Four patients had tumours located within the inferior vena cava, and five had tumours that involved the right heart. Seven patients with residual uterus myoma showed enhanced heterogeneous contrast of the uterus. Eight tumours extended into the inferior vena cava through the iliac veins and one through the bilateral iliac veins. All the IVLs demonstrated heterogeneous enhancement on contrast-enhanced CT and appeared to have lower density than the contrast-enhanced venous blood. On the coronal and sagittal images, tumours were continuous and, when the right heart was involved, the lesions displayed a characteristic "walking stick head" or "snake head" appearance. The lesion looked similar to a "sieve" on axial contrast-enhanced CT images, and a "luffa sponge" on post-processing coronal or sagittal images. CONCLUSION: Contrast-enhanced CT imaging has unique advantages in the diagnosis of IVL, namely, luffa sponge and sieve appearance, which can be helpful for differential diagnosis. Contrast-enhanced CT can demonstrate tumour location and full-scale extension pathway, which are important for diagnosis, surgical planning, and follow-up of the tumour.

[The role and significance of digital reconstruction technique in liver segments based on portal vein structure].

Objective: To study the segment of liver according to the large amount of three-dimensional(3D) reconstructive images of normal human livers and the vascular system, and to recognize the basic functional liver unit based on the anatomic features of the intrahepatic portal veins. Methods: The enhanced CT primitive DICOM files of 1 260 normal human livers from different age groups who treated from October 2013 to February 2017 provided by 16 hospitals were analyzed using the computer-aided surgery system.The 3D liver and liver vascular system were reconstructed, and the digital liver 3D model was established.The vascular morphology, anatomical features, and anatomical distributions of intrahepatic portal veins were statistically analyzed. Results: The digital liver model obtained from the 3D reconstruction of CAS displayed clear intrahepatic portal vein vessels of level four.Perform a digital liver segments study based on the analysis of level four vascular distribution areas.As the less anatomical variation of left hepatic portal vein, the liver was classified into four types of liver segmentation mainly based on right hepatic portal vein.Type A was similar to Couinaud or Cho's segmentation, containing 8 segments(537 cases, 42.62%). Type B contained 9 segments as there are three ramifications of right-anterior portal vein(464 cases, 36.82%). The main difference for Type C was the variation of right-posterior portal vein which was sector shape(102 cases, 8.10%). Type D contained the cases with special portal vein variations, which needs three-dimensional simulation to design individualized liver resection plan(157 cases, 12.46%). These results showed that there was no significant difference in liver segmental typing between genders(χ(2)=2.179, P=0.536) and did not reveal any significant difference in liver segmental typing among the different age groups(χ(2)=0.357, P=0.949). Conclusions: The 3D digital liver model can demonstrate the true 3D anatomical structures, and its spatial vascular variations.The observation of anatomic features, distribution areas of intrahepatic portal veins and individualized liver segmentation achieved via digital medical 3D visualization technology is of great value for understand the complexity of liver anatomy and to guide the precise hepatectomy.

Molecular cloning and expression analysis of a fish specific interferon regulatory factor, IRF11, in orange spotted grouper, Epinephelus coioides.

Interferon regulatory factors (IRFs) are transcription mediators which play vital roles in multiple biological processes, such as antiviral defense, immune response, cell growth regulation and apoptosis. A fish specific IRF, termed IRF11, has been identified in previous study through searching fish genome databases. Herein, a transcript of IRF11, EcIRF11 was cloned from orange-spotted grouper, Epinephelus coioides. The EcIRF11 cDNA sequence has 1573 bp in length, encoding a putative protein of 261 amino acids, with a high degree of similarity found between EcIRF11 and its teleost counterparts. Comparative analyses in teleost genomes revealed that IRF11 may have an ancient origin at least 450 million years ago, and the locus harbouring IRF11 might have experienced chromosomal rearrangement and/or inversion during evolution. Expression analysis revealed that the other two members, IRF1 and IRF2 also in the IRF1 subgroup (SG) as IRF11, exhibited high expression levels in early experimental infection phase in response to viral stimulation of poly I:C and to bacterial stimulation of Vibrio parahaemolyticus infections in the fish, while EcIRF11 is not transcriptionally modulated at the examined time points except in kidney at 6 h following poly I:C stimulation. Taken together, the results obtained in this study indicate that IRF11 might have been originated from the same ancestor as IRF1 and IRF2, but exhibits distinct basal and induced expression, implying its different function which needs further characterization.

TBK1-like transcript negatively regulates the production of IFN and IFN-stimulated genes through RLRs-MAVS-TBK1 pathway.

TANK-binding kinase 1 (TBK1) is an essential serine/threonine-protein kinase required for Toll-like receptor (TLR)- and retinoic acid-inducible gene I (RIG-I) -mediated induction of type I IFN and host antiviral defense. In the present study, TBK1-like transcript, namely TBK1L, was cloned from zebrafish. Compared with TBK1, TBK1L contains an incomplete S_TKc domain, and lacks UBL_TBK1_like domain. Realtime PCR showed that TBK1L was constitutively produced in embryos, early larvae and ZF4 cells, and unchanged in ZF4 cells following SVCV infection. Overexpression of TBK1 but not TBK1L resulted in significant activation of zebrafish IFN1 and IFN3 promoters. Similarly, TBK1L had little impact on the antiviral state of the cells. However, the overexpression of TBK1L negatively regulated the induction of zebrafish IFN1 and/or IFN3 promoters mediated by the retinoic acid-inducible gene I-like receptors (RLRs), MAVS and TBK1. In addition, the overexpression of TBK1L in zebrafish embryos led to the decreased production of many IFN-stimulated genes induced by TBK1. Collectively, these data support that zebrafish TBK1L negatively regulates RLRs-MAVS-TBK1 pathway.

MRI findings in intraspinal mature teratoma.

AIM: To characterise and evaluate magnetic resonance imaging (MRI) images for their clinical value in diagnosing and assessing intraspinal mature teratoma. MATERIALS AND METHODS: MRI images obtained from eight patients with a histopathologically verified intraspinal mature teratoma were analysed retrospectively regarding tumour location, size, and margins. Additionally, the signal intensity and enhancement pattern on MRI and other associated malformations were also assessed. RESULTS: Three cases that contained fatty tissue showed markedly heterogeneous hyperintense signalling on T1-weighted images, and mixed hyperintense and hypointense signalling on T2-weighted images and fat-suppression sequences. All three of those cases showed an irregular peripheral fatty tissue signal, and one case showed additional patches of an interspersed calcification signal. The remaining five cases without fatty tissue displayed heterogeneous hyperintense signalling on T1-weighted images and T2-weighted images, and also on fat-suppression sequences. Four of the five cases showed additional patches of interspersed nodular calcification signals. Contrast-enhanced MRI images showed only slight enhancement (n=3). CONCLUSIONS: MRI is regarded as the reference standard diagnostic technique to reveal the location of teratomas and the degree of spinal cord involvement. In most cases, MRI provides accurate anatomical and histological information, which is necessary for patients with suspected intraspinal mature teratoma.

Interferon regulatory factor 10 (IRF10): Cloning in orange spotted grouper, Epinephelus coioides, and evolutionary analysis in vertebrates.

IRF10 gene was cloned in orange spotted grouper, Epinephelus coioides, and its expression was examined following poly(I:C) stimulation and bacterial infection. The cDNA sequence of grouper IRF10 contains an open reading frame of 1197 bp, flanked by 99 bp 5'-untranslated region and 480 bp 3'- untranslated region. Multiple alignments showed that the grouper IRF10 has a highly conserved DNA binding domain in the N terminus with characteristic motif containing five tryptophan residues. Quantitative real-time PCR analysis revealed that the expression of IRF10 was responsive to both poly(I:C) stimulation and Vibrio parahemolyticus infection, with a higher increase to poly(I:C), indicating an important role of IRF10 in host immune response during infection. A phyletic distribution of IRF members was also examined in vertebrates, and IRF10 was found in most lineages of vertebrates, not in modern primates and rodents. It is suggested that the first divergence of IRF members might have occurred before the evolutionary split of vertebrate and cephalochordates, producing ancestors of IRF (1/2/11) and IRF (4/8/9/10)[(3/7) (5/6)], and that the second and/or third divergence of IRF members occurred following the split, thus leading to the subsets of the IRF family in vertebrates.

Cloning and expression analyses of interferon regulatory factor (IRF) 3 and 7 genes in European eel, Anguilla anguilla with the identification of genes involved in IFN production.

Interferon regulatory factor (IRF) 3 and IRF7 have been identified as regulators of type I interferon (IFN) gene expression in mammals. In the present study, the two genes were cloned and characterized in the European eel, Anguilla anguilla. The full-length cDNA sequence of IRF3 and IRF7 in the European eel, named as AaIRF3 and AaIRF7 consists of 2879 and 2419 bp respectively. Multiple alignments showed that the two IRFs have a highly conserved DNA binding domain (DBD) in the N terminus, with the characteristic motif containing five tryptophan residues, which is a feature present in their mammalian homologues. But, IRF7 has only four of the five residues in other species of fish. The expression of AaIRF3 and AaIRF7 both displayed an obvious dose-dependent manner following polyinosinic:polycytidylic acid (PolyI:C) challenge. In vivo expression analysis showed that the mRNA level of AaIRF3 and AaIRF7 was significantly up-regulated in response to PolyI:C stimulation in all examined tissues/organs except in muscle, with a lower level of increase observed in response to lipopolysaccharide (LPS) challenge and Edwardsiella tarda infection, indicating that AaIRF3 and AaIRF7 may be more likely involved in antiviral immune response. In addition, some pattern recognition receptors genes related with the production of type I IFNs and those genes in response to type I IFNs were identified in the European eel genome database, indicating a relatively conserved system in the production of type I IFN and its signalling in the European eel.

Complementary DNA sequences of the constant regions of T-cell antigen receptors α, ß and γ in mandarin fish, Siniperca chuatsi Basilewsky, and their transcriptional changes after stimulation with Flavobacterium columnare.

In this study, the constant-region genes (Cα, Cß and Cγ) that encode the T-cell antigen receptor (TCR) α, ß and γ chains were cloned from mandarin fish, Siniperca chuatsi Basilewsky, an important freshwater fish species in China. The complementary DNA sequences of Cα, Cß and Cγ were 843, 716 and 906 base pairs (bp) in length and had a 465-, 289- and 360-bp 3' untranslated region, encoding 125, 142 and 182 amino acids, respectively. The amino-acid sequences of the constant regions of mandarin fish TCR α, ß and γ chains (encoded by Cα, Cß and Cγ, respectively) were most similar to those of their teleost counterparts, showing 60% similarity with pufferfish, 48% similarity with Atlantic salmon and 57% similarity with flounder, respectively. The phylogenetic analysis revealed that the mandarin fish Cα, Cß and Cγ were clustered, respectively, with their vertebrate counterparts. The mandarin fish Cα, Cß and Cγ could also be separated into four domains: immunoglobulin; connecting peptide (CP); transmembrane (TM); and cytoplasmic tail. Several conserved features in mammalian TCRs were also found in those of mandarin fish, such as a conserved cysteine residue in the CP domain of Cα, necessary for creating an interchain disulphide bond with the TCR ß chain, and a conserved antigen receptor TM motif in Cα and Cß. Meanwhile, transcripts of Cα, Cß and Cγ were detectable in all examined organs, with a stronger signal observed in lymphoid organs. In addition, the temporal transcriptional changes for Cα and Cγ were investigated, 1, 2, 3, 4, 5, 6 and 8 weeks after stimulation with Flavobacterium columnare, in head kidney, spleen, blood, thymus, gill and intestine, using real-time polymerase chain reaction. The results demonstrated stimulation-dependent up-regulations in almost all tissues examined, which indicates that T cells may play important roles in preventing mandarin fish from bacterial invasion. In particular, apart from thymus, T cells were distributed mainly in gill and intestine, where striking up-regulation of Cγ was also observed. These results will facilitate functional studies of teleost TCRs and T cells.