This paper reports the cloning and sequencing of interleukin (IL)-22 in two gadoid fish, cod (Gadus morhua) and haddock (Melanogrammus aeglefinus). The complete transcript of this gene was 1002 and 1154 bp respectively, of which 492 bp was the open reading frame (ORF) in both genes. High amino acid identity (88.3%) was found between these genes but was less than 50% aa identity to other known genes. The gene organisation of haddock IL-22 consisted of five exons and four introns, as with other IL-10 family members. Expression studies showed that IL-22 is constitutively expressed in gill, with low level expression also observed in gut, gonad and head kidney. In a vaccination experiment, haddock were injected intraperitoneally with formalin-killed Vibrio anguillarum or with PBS, and 2 months later challenged by immersion in 10(7)cfu/ml V. anguillarum for 30 min. Head kidney and gill samples were collected prior to challenge and 24, 48 and 72 h post-challenge (hpc) for Real-time PCR analysis of IL-22 expression. No significant changes in IL-22 expression were observed in head kidney tissue but vaccinated fish showed a significantly increased expression of IL-22 24 hpc in gill and no mortalities were seen in these fish. In contrast, control fish, which started to succumb to the disease from 72 hpc, showed no significant increase in gill expression after challenge.

An initial bioinformatics investigation followed by cloning and sequencing analysis, has led to the identification of three novel members (omDB-2, omDB-3, omBD-4) of the beta-defensin family in rainbow trout (Oncorhynchus mykiss). The contiguous sequences could be translated to give predicted peptides of 62 (omDB-2), 63 (omDB-3) and 68 (omDB-4) amino acids (aa) in length, with mature peptides of 43 (omDB-2), 39 (omDB-3) and 42 (omDB-4) aa, with no obvious proregion present. Analysis of the gene organization found that all three new genes contained three exons divided by two introns, as seen in defensin genes of other fish species. Constitutive expression of all the trout defensins was detected by RT-PCR in a wide range of mucosal and systemic tissues from healthy fish, with omDB-3 and omDB-4 showing the highest expression levels. Following bacterial challenge in vivo, the defensin genes were induced at the three mucosal sites examined (skin, gill, gut), with levels of omDB-2 and omDB-3 increased some 16-fold in gut and gill respectively. Using polyinosinic polycytosinic RNA (polyI:C) as a viral mimic, all of the four trout beta-defensin genes were induced in head kidney primary leucocyte cultures at 4h post-stimulation, with omDB-1 and omDB-3 particularly highly expressed. These data suggest that beta-defensins are likely an important component of the innate defences of fish, and reveal an added level of antimicrobial peptide complexity in fish to that known previously.

Interferons (IFNs) are the hallmark of the vertebrate antiviral system. Two of the three IFN families identified in higher vertebrates are now known to be important for antiviral defence in teleost fish. Based on the cysteine patterns, the fish type I IFN family can be divided into two subfamilies, which possibly interact with distinct receptors for signalling. The fish type II IFN family consists of two members, IFN-γ with similar functions to mammalian IFN-γ and a teleost specific IFN-γ related (IFN-γrel) molecule whose functions are not fully elucidated. These two type II IFNs also appear to bind to distinct receptors to exert their functions. It has become clear that fish IFN responses are mediated by the host pattern recognition receptors and an array of transcription factors including the IFN regulatory factors, the Jak/Stat proteins and the suppressor of cytokine signalling (SOCS) molecules.

This study investigated the effects of long-term simulated weightlessness on liver morphology, enzymes, glycogen, and apoptosis related proteins by using two-month rat-tail suspension model (TS), and liver injury improvement by rat-tail suspension with resistance training model (TS&RT). Microscopically the livers of TS rats showed massive granular degeneration, chronic inflammation, and portal fibrosis. Mitochondrial and endoplasmic reticulum swelling and loss of membrane integrity were observed by transmission electron microscopy (TEM). The similar, but milder, morphological changes were observed in the livers of TS&RT rats. Serum biochemistry analysis revealed that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly higher (p<0.05) in TS rats than in controls. The levels of ALT and AST in TS&RT rats were slightly lower than in RT rats, but they were insignificantly higher than in controls. However, both TS and TS&RT rats had significantly lower levels (p<0.05) of serum glucose and hepatic glycogen than in controls. Immunohistochemistry demonstrated that the expressions of Bax, Bcl-2, and active caspase-3 were higher in TS rats than in TS&RT and control rats. Real-time polymerase chain reaction (real-time PCR) showed that TS rats had higher mRNA levels (P < 0.05) of glucose-regulated protein 78 (GRP78) and caspase-12 transcription than in control rats; whereas mRNA expressions of C/EBP homologous protein (CHOP) and c-Jun N-terminal kinase (JNK) were slightly higher in TS rats. TS&RT rats showed no significant differences of above 4 mRNAs compared with the control group. Our results demonstrated that long-term weightlessness caused hepatic injury, and may trigger hepatic apoptosis. Resistance training slightly improved hepatic damage.

The interleukin (IL)-10 cytokine family includes IL-10, IL-19, IL-20, IL-22, IL-24, IL-26 and the lambda/type III interferons. They are highly pleiotropic and mediate a variety of activities, including immune suppression and antibacterial immunity. To exert their functions they signal through a heterodimeric receptor composed of a subunit with a long intracellular domain (R1 type receptors; IL-10R1, IL-20R1 or IL-22R1) and a subunit with a short intracellular domain (R2 type receptors; IL-10R2 or IL-20R2). In this study we report the identification of three R1 type receptors (named IL-10R1/CRFB7, IL-20R1a/CRFB8a and IL-20R1b/CRFB8b) and one R2 type receptor (named IL-10R2/CRFB4) in rainbow trout. The nomenclature of the receptors was supported by homology analysis, conserved motifs and phylogenetic tree analysis, confirming they belong to the piscine class 2 cytokine receptor family. For instance, they all displayed the presence of characteristic features, such as conserved fibronectin type-III domains. Expression analysis in tissues collected from healthy fish revealed different patterns of expression for each receptor, suggesting their potential involvement in different types of immune responses. When studying the modulation of the genes in cell lines and primary cultures, a greater effect was observed in the cell lines, where the expression of most receptors was affected by incubation with microbial mimics (LPS and PolyI:C) or the pro-inflammatory cytokine rIFN-γ. In addition, expression of the four receptors was modulated by viral infection, suggesting a potential involvement of such receptors and their ligands in antiviral defence.

Pre-diabetes and non-alcoholic fatty liver disease (NAFLD) are associated with an unhealthy lifestyle and pose extremely high costs to the healthcare system. In this study, we aim to explore whether individualized aerobic exercise (AEx) and low carbohydrate diet (LCh) intervention affect hepatic fat content (HFC) in pre-diabetes via modification of gut microbiota composition and other post-interventional effects.

The early stages of the immune response are regulated by key cytokines including both interleukin 1beta (IL-1beta) and interferon-gamma (IFN-gamma) which stimulate panels of responsive genes via conserved signal transduction pathways. To further our understanding of the transcriptional response to these cytokines in lower vertebrates we have utilized microarray analysis to characterize the transcriptional response to recombinant rainbow trout IL-1beta and IFN-gamma in the trout macrophage cell line RTS-11.

CD4 is a transmembrane glycoprotein fundamental for cell-mediated immunity. Its action as a T cell co-receptor increases the avidity of association between a T cell and an antigen-presenting cell by interacting with portions of the complex between MHC class II and TR molecules. In this paper we report the cDNA cloning, expression and structural analysis of a CD4 homologue from sea bass (Dicentrarchus labrax). The sea bass CD4 cDNA consists of 2071 bp that translates in one reading frame to give the entire molecule containing 480 amino acids. The analysis of the sequence shows the presence of four putative Ig-like domains and that some fundamental structural features, like a disulphide bond in domain D2 and the CXC signalling motif in the cytoplasmic tail, are conserved from sea bass to mammals. Real-time PCR analysis showed that very high levels of CD4 mRNA transcripts are present in thymus, followed by gut and gills. In vitro stimulation of head kidney leukocytes with LPS and PHA-L gave an increase of CD4 mRNA levels after 4h and a decrease after 24h. Homology modelling has been applied to create a 3D model of sea bass CD4 and to investigate its interaction with sea bass MHC-II. The analysis of the 3D complex between sea bass CD4 and sea bass MHC-II suggests that the absence of a disulfide bond in the CD4 D1 domain could make this molecule more flexible, inducing a different conformation and affecting the binding and the way of interaction between CD4 and MHC-II. Our results will add new insights into the sea bass T cell immune responses and will help in the identification of T cell subsets in teleost fishes to better understand the evolution of cell-mediated immunity from fish to mammals.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is an autosomal recessive disorder of fatty acid, amino acid, and choline metabolism caused by mutations in EFTA, EFTB, or ETFDH. Many MADD patients are responsive to treatment with riboflavin, termed riboflavin-responsive MADD (RR-MADD). Here, we report three novel mutations and one previously reported mutation in ETFDH in four RR-MADD patients who presented at various ages, and characterize the corresponding changes in ETF-QO protein structure. Clinicians should consider MADD in the differential diagnosis when patients present with muscle weakness and biochemical abnormalities. Gene testing plays a critical role in confirming the diagnosis of MADD, and may not only prevent patients from invasive testing, but also allow timely initiation of riboflavin treatment. The novel variants in ETFDH and the corresponding clinical features reported here enrich the allelic heterogeneity of RR-MADD and provide insight into genotype-phenotype relationships.

Prolongation of QRS duration in electrocardiogram is one of the risk factors for morbidity and mortality in many kinds of cardiac diseases. However, its molecular mechanism is unknown. In this study, utilizing experimental autoimmune myocarditis (EAM) as a disease model, we show that the prolongation of QRS duration is accompanied by elevated phosphorylation of connexin 43 (Cx43) at Ser368 (pS368 Cx43). In cultured cells, inflammatory cytokine IL-1β activates p38 MAPK to up-regulate pS368 Cx43 and impairs cell-to-cell communication. In isolated hearts of normal rats, perfusion of IL-1β not only increases pS368 Cx43 but also impairs cell-to-cell communication and prolongs QRS duration. Furthermore, blockade of p38 MAPK down-regulates pS368 Cx43, improves cell-to-cell communication and reduces QRS duration in EAM. These findings suggest that up-regulation of pS368 Cx43 by IL-1β via p38 MAPK contributes to the prolongation of QRS duration and could be a therapeutic target for myocarditis-induced prolongation of QRS duration.

Objective: To assist clinicians to operate repetitive Transcranial Magnetic Stimulation (rTMS) precisely based on the coil magnetic field spatial distribution with Magnetic Resonance Imaging (MRI) Navigation. Methods: A fast method for calculating electromagnetic fields in layered brain structures in frequency domain was proposed. By approaching Bessel function in different intervals, the integral with a highly oscillatory kernel was transformed into two parts: a definite integral and a weakened oscillatory one. The distribution of induced current density and magnetic field intensity of rTMS stimulation effect on brain was quantitatively calculated, so that clinicians could intuitively grasp the safe range of coil stimulation on the brain. Then, the crucial factor of the stimulation effect of rTMS was determined, and an accurate coil positioning of the rTMS efficiently was completed. Result: The maximal attenuation of induced electric field and magnetic induction intensity was 72.20 and 86.867% at 3 cm away from the skin in the brain layered model. The clinical examination results of electric field intensity distribution, magnetic field intensity distribution, current density distribution, layered brain modeling, and coil location speed in the brain model teaching group were significantly higher than those in the traditional teaching group (P < 0.001). Conclusion: It is suitable for clinicians to quickly complete the precise positioning of rTMS, master the adjustment of coil stimulation therapeutic parameters, and realize the precise positioning operation of rTMS with MRI navigation in intracranial. Clinical Trial registration: Chinese Clinical Trial Registry (ChiCTR1800018616); Registered on 30th September 2018.

The interleukin (IL) -1 family members play an important role in regulating inflammatory responses and their functions are mediated by a group of receptors consisting of immunoglobulin and Toll/IL-1 receptor (TIR) domains. In humans, 10 IL-1Rs are found. In this study, 5 IL-1 receptors including IL-1R3/IL-1RAcP, IL-1R8/SIGIRR, IL-1R9a/IL-1RAcPL1a, IL-1R9b/IL-1RAcPL1b and IL-1R10/IL-1RAcPL2 were identified in grass carp (Ctenopharyngodon idella). Phylogenetic analysis reveals that the IL-1R9a/IL-1RAcPL1a and IL-1R9b/IL-1RAcPL1b share significantly high sequence similarity and are believed to have been duplicated from the same gene prior to the radiation of teleosts. Further, these two receptors closely relate to the IL-1R10/IL-1RAcPL2, suggesting that they may have evolved from a common ancestor. The IL-1R3/IL-1RAcP, IL-1R9a/IL-1RAcPL1a, IL-1R9b/IL-1RAcPL1b and IL-1R10/IL-1RAcPL2 are highly expressed in the brain. Stimulation of primary spleen leucocytes by LPS and intraperitoneal injection of fish with poly (I:C) or bacterial infection results in significant increases of IL-1R3/IL-1RAcP expression. Interestingly, the IL-1R8/SIGIRR and IL-1R10/IL-1RAcPL2 showed similar expression patterns.

Imbalance of osteoblast and osteoclast in adult leads to a variety of bone-related diseases, including osteoporosis. Thus, suppressing the activity of osteoclastic bone resorption becomes the main therapeutic strategy for osteoporosis. Asperpyrone A is a natural compound isolated from Aspergillus niger with various biological activities of antitumour, antimicrobial and antioxidant. The present study was designed to investigate the effects of Asperpyrone A on osteoclastogenesis and to explore its underlining mechanism. We found that Asperpyrone A inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner when the concentration reached 1 µm, and with no cytotoxicity until the concentration reached to 10 µm. In addition, Asperpyrone A down-regulated the mRNA and protein expression of NFATc1, c-fos and V-ATPase-d2, as well as the mRNA expression of TRAcP and Ctsk. Furthermore, Asperpyrone A strongly attenuated the RNAKL-induced intracellular Ca2+ oscillations and ROS (reactive oxygen species) production in the process of osteoclastogenesis and suppressed the activation of MAPK and NF-κB signalling pathways. Collectively, Asperpyrone A attenuates RANKL-induced osteoclast formation via suppressing NFATc1, Ca2+ signalling and oxidative stress, as well as MAPK and NF-κB signalling pathways, indicating that this compound may become a potential candidate drug for the prevention or treatment of osteoporosis.

Purpose: The purpose of this systematic review and meta-analysis was to evaluate the effects of massage on alleviating delayed onset of muscle soreness (DOMS) and muscle performance after strenuous exercise. Method: Seven databases consisting of PubMed, Embase, EBSCO, Cochrane Library, Web of Science, CNKI and Wanfang were searched up to December 2016. Randomized controlled trials (RCTs) were eligible and the outcomes of muscle soreness, performance (including muscle maximal isometric force (MIF) and peak torque) and creatine kinase (CK) were used to assess the effectiveness of massage intervention on DOMS. Results: Eleven articles with a total of 23 data points (involving 504 participants) satisfied the inclusion criteria and were pooled in the meta-analysis. The findings demonstrated that muscle soreness rating decreased significantly when the participants received massage intervention compared with no intervention at 24 h (SMD: -0.61, 95% CI: -1.17 to -0.05, P = 0.03), 48 h (SMD: -1.51, 95% CI: -2.24 to -0.77, P < 0.001), 72 h (SMD: -1.46, 95% CI: -2.59 to -0.33, P = 0.01) and in total (SMD: -1.16, 95% CI: -1.60 to -0.72, P < 0.001) after intense exercise. Additionally, massage therapy improved MIF (SMD: 0.56, 95% CI: 0.21-0.90, P = 0.002) and peak torque (SMD: 0.38, 95% CI: 0.04-0.71, P = 0.03) as total effects. Furthermore, the serum CK level was reduced when participants received massage intervention (SMD: -0.64, 95% CI: -1.04 to -0.25, P = 0.001). Conclusion: The current evidence suggests that massage therapy after strenuous exercise could be effective for alleviating DOMS and improving muscle performance.

Objective To investigate changes in nucleus pulposus cell expression and secretion of interleukin (IL)-1β and tumour necrosis factor (TNF)-α following stimulation with a low-frequency (LF) pulsed electromagnetic field (PEMF). Methods Primary rat nucleus pulposus cells were isolated and cultured in vitro, followed by stimulation with LF-PEMFs at a frequency of 2 Hz and different intensities, ranging from 0.5-3.0 A/m. Cells were observed for morphological changes, and proliferation rates were measured by cell viability counts. Expression of IL-1β and TNF-α within the nucleus pulposus cells was measured using western blotting, and levels of IL-1β and TNF-α secreted in the culture media were measured using enzyme-linked immunosorbent assay. Results Stimulation of nucleus pulposus cells with LF-PEMFs did not appear to affect cell morphology or nucleus pulposus cell IL-1β and TNF-α expression levels. LF-PEMFs did not significantly affect cell proliferation, however, levels of IL-1β and TNF-α secreted into the culture media were found to be significantly reduced in an intensity-dependent manner. Conclusion Low-frequency PEMF stimulation may inhibit secretion of IL-1β and TNF-α in cultured nucleus pulposus cells.

As an increasing number of plant genome sequences become available, it is clear that gene content varies between individuals, and the challenge arises to predict the gene content of a species. However, genome comparison is often confounded by variation in assembly and annotation. Differentiating between true gene absence and variation in assembly or annotation is essential for the accurate identification of conserved and variable genes in a species. Here, we present the de novo assembly of the B. napus cultivar Tapidor and comparison with an improved assembly of the Brassica napus cultivar Darmor-bzh. Both cultivars were annotated using the same method to allow comparison of gene content. We identified genes unique to each cultivar and differentiate these from artefacts due to variation in the assembly and annotation. We demonstrate that using a common annotation pipeline can result in different gene predictions, even for closely related cultivars, and repeat regions which collapse during assembly impact whole genome comparison. After accounting for differences in assembly and annotation, we demonstrate that the genome of Darmor-bzh contains a greater number of genes than the genome of Tapidor. Our results are the first step towards comparison of the true differences between B. napus genomes and highlight the potential sources of error in future production of a B. napus pangenome.

Nuclear factor of activated T cells 3 (NFATc3) has been reported to upregulate type I interferons (IFNs) expression, and the abnormal expression and activation of NFATc3 were closely related to tumorigenesis. However, the potential function of NFATc3 in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) remains to be elucidated. In this study, we found that NFATc3 gene was frequently deleted and downregulated in HCC tumor tissues, and that the downregulation of NFATc3 was associated with poor prognosis of HCC patients. The gain- and loss-of-function experiments demonstrated that NFATc3 inhibited HCC cell proliferation and invasion, as well as HBV replication. Mechanistically, NFATc3 could bind to the promoters of IFNL1 and IFNB1 genes and prompt the production of IFNs and interferon-stimulated genes. Furthermore, retinoic acid-inducible gene-I (RIG-I) pathway activation increased NFATc3 expression and nuclear localization, and activated NFATc3 further enhanced RIG-I-mediated IFN responses. Collectively, our findings reveal a novel regulatory signaling cascade, the RIG-I/NFATc3/IFNs axis, which inhibits hepatocarcinogenesis and HBV replication by enhancing the immune response in hepatocytes, and this functional axis might potentially be exploited for therapeutic benefits in the clinical treatment of HBV-related HCC.

Colorectal cancer (CRC) is the third most common cancer in the world known for its poor recurrence-free prognosis. Previous studies have shown that it is closely linked with cancer stem cells (CSCs), which have self-renewal potential and the capacity to differentiate into diverse populations. Nanog is an important transcription factor that functions to maintain the self-renewal and proliferation of embryonic stem cells; however, many recent studies have shown that Nanog is also highly expressed in many cancer stem cells. To investigate whether Nanog plays a crucial role in maintaining the stemness of colorectal CSCs, RNA interference was used to downregulate Nanog expression in the CRC stem cell line, EpCAM+CD44+HCT-116 cells (CCSCs). We examined the anti-tumor function of Nanog in vitro and in vivo, using small interfering RNA. Our results revealed that the Nanog mRNA expression level in CCSCs was higher than that in HCT-116 cells. We found that the depletion of Nanog inhibited proliferation and promoted apoptosis in CCSCs. In addition, the invasive ability of CCSCs was markedly restricted when Nanog was silenced by small interfering RNA. Furthermore, we found that the silencing of Nanog decreased tumor size and weight and improved the survival rate of tumor-bearing mice. In conclusion, these findings collectively demonstrate that Nanog, which is highly expressed in CRC stem cells, is a key factor in the development of tumor growth, and it may serve as a potential marker of prognosis and a novel and effective therapeutic target for the treatment of CRC.

Tissue engineering is a promising strategy for the repair of large-scale bone defects, in which scaffolds and growth factors are two critical issues influencing the efficacy of bone regeneration. Unfortunately, the broad application of growth factors is limited by their poor stability in the scaffolds. In the present study, the strictly controlled expression of human bone morphogenetic protein-4 (hBMP-4) in the presence of doxycycline is achieved by adding an hBMP-4 gene fragment into a non-viral artificial restructuring plasmid vector (pSTAR) to form the pSTAR-hBMP-4 plasmid (phBMP-4). Furthermore, the controlled release of phBMP-4 is obtained with an electroactive tissue engineering scaffold, generated by combining a triblock copolymer of poly(l-lactic acid)-block-aniline pentamer-block-poly(l-lactic acid) (PLA-AP) with poly(lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA). This PLGA/HA/PLA-AP/phBMP-4 composite scaffold, with controlled gene release and Dox-regulated gene expression upon electrical stimulation, operating synergistically, exhibits an improved cell proliferation ability, enhanced osteogenesis differentiation in vitro, and effective bone healing in vivo in a rabbit radial defect model. Taking these results together, the proposed smart PLGA/HA/PLA-AP/phBMP-4 scaffold lays a solid theoretical and experimental basis for future applications of such multi-functional materials in bone tissue engineering to help patients in need.

Cancer cachexia is a cancer-induced multifactorial debilitating syndrome directly accounting for 20% of cancer deaths without effective therapeutic approaches. It is extremely urgent to explore effective anti-cachexia drugs to ameliorate muscle and fat loss in cachexia patients.