Lipopolysaccharide-induced TNF-α Factor Research Articles

The P4' Peptide-Carrying Bacillus subtilis in Cottonseed Meal Improves the Chinese Mitten Crab Eriocheir sinensis Innate Immunity, Redox Defense, and Growth Performance.

This study developed a recombinant Bacillus subtilis to carry the LGSPDVIVIR peptide (cmP4) isolated from the hydrolyzed products of cottonseed meal with excellent antioxidant and immune-enhancing properties in vitro. It was carried as a tandem of five cmP4 peptides (cmP4') to be stably expressed on a large scale. Then, its effectiveness was evaluated in Chinese mitten crab (Eriocheir sinensis) based on growth performance, redox defense, and innate immunity. A total of 280 crabs (mean body weight: 41.40 ± 0.14) were randomly assigned to seven diets including a control one (without B. subtilis) and six experimental ones with different doses (107,108, and 109 CFU/kg) of unmodified and recombinant B. subtilis, respectively, for 12 weeks. Each diet was tested in four tanks of crabs (10/tank). In terms of bacterial dosages, the final weight (FW), weight gain (WG), hemolymph and hepatopancreatic activities of superoxide dismutase (SOD), catalase (CAT), lysosome (LZM), acid phosphatase (ACP) and alkaline phosphatase (AKP), and hepatopancreatic transcriptions of cat, mitochondrial manganese superoxide dismutase (mtmnsod), thioredoxin-1 (trx1), and prophenoloxidase (propo) all increased significantly with increasing B. subtilis dosages, while hemolymph and hepatopancreatic malondialdehyde (MDA) content and the transcriptions of toll like receptors (tlrs), NF-κB-like transcription factor (relish), and lipopolysaccharide-induced TNF-α factor (litaf) all decreased remarkably. In terms of bacterial species, the recombinant B. subtilis group obtained significantly high values of FW, WG, hemolymph, and hepatopancreatic activities of SOD, CAT, LZM, ACP, and AKP, and the transcriptions of mtmnsod, peroxiredoxin 6 (prx6), and propo compared with the unmodified B. subtilis, while opposite results were noted in hemolymph and hepatopancreatic MDA content and the transcriptions of tlrs, relish, and litaf. These results indicated that dietary supplementation with 109 CFU/kg of recombinant B. subtilis can improve the growth performance, redox defense, and nonspecific immunity of E. sinensis.

SpTNF regulates apoptosis and antimicrobial peptide synthesis in mud crab (Scylla paramamosain) during white spot syndrome virus infection.

Tumor necrosis factor (TNF) is an inflammatory cytokine that is important in cell survival, proliferation, differentiation, and death. However, the functions of TNF in the immune responses of invertebrates have been less studied. In this study, SpTNF was cloned and characterized from mud crab (Scylla paramamosain) for the first time. SpTNF contains an open reading frame of 354 bp encoding 117 deduced amino acids, with a conserved C-terminal TNF homology domain (THD) domain. RNAi knockdown of SpTNF reduced hemocyte apoptosis and antimicrobial peptide (AMP) synthesis. Expression of SpTNF was initially down-regulated but subsequently up-regulated after 48 h in hemocytes of mud crabs after WSSV infection. Results of RNAi knockdown and overexpression showed that SpTNF inhibits the WSSV infection through activating apoptosis, NF-κB pathway, and AMP synthesis. Furthermore, the lipopolysaccharide-induced TNF-α factor (SpLITAF) can regulate the expression of SpTNF, induction of apoptosis, and activation of the NF-κB pathway and AMP synthesis. The expression and nuclear translocation of SpLITAF were regulated by WSSV infection. SpLITAF knockdown increased the WSSV copy number and VP28 gene expression. Taken together, these results proved the protective function of SpTNF, which is regulated by SpLITAF, in the immune response of mud crabs against WSSV through the regulation of apoptosis and activation of AMP synthesis.

Pathologic characterization of white striping myopathy in broiler chickens

White striping (WS) is an emerging myopathy of broiler chickens characterized by white striation of muscle. Despite the recent advances, the pathomechanism underlying the WS remains elusive.The aim of this study was to characterize morphological and molecular features of WS in broiler chickens. 50 pectoralis muscles were collected from 55 days old ROSS 308 broiler chickens with a mean weight of 3.5 kg. Samples were snap frozen and analyzed by histopathology, immunohistochemistry, and immunofluorescence. Real-time-PCR was used to evaluate the expression of different cytokines.Histological lesions were observed in all examined animals, both with and without macroscopic evidence of WS. WS muscles showed endomysial and perivascular inflammatory infiltrates of macrophages and cluster of differentiation (CD)8-positive T lymphocytes with severe myofiber atrophy, necrosis, fibrosis and replacement by adipose tissue. There was diffuse sarcoplasmic and sarcolemmal overexpression of the major histocompatibility complex class I (MHC I). The severity of the histologic lesions was positively correlated with the macroscopic degree of white striations. IL-6, IL-17 and lipopolysaccharide-induced TNF-α factor (LITAF) were overexpressed in severe lesions of WS. The presence of the CD8/MHC I complexes, together with the higher expression of IL-6, IL-17 and LITAF in severe degree of WS, suggest that the immune response may be involved in the progression of this myopathy and can be consistent with a hypoxia-induced inflammatory myopathy. These results help to understand the pathomechanism of WS contributing to the reduction of economic losses and improving poultry welfare.

Lipopolysaccharide-induced TNFα factor (LITAF) promotes inflammatory responses and activates apoptosis in zebrafish Danio rerio.

Lipopolysaccharide-induced TNFα factor (LITAF) is an important transcription factor which activates the transcription of TNFα and regulates cell apoptosis and inflammatory response. In the present study, a LITAF gene homologue was identified in zebrafish (Danio rerio) and was shown to be well conserved in the protein sequence, genomic organization and synteny with human LITAF. DrLITAF was constitutively expressed in tissues, with the highest expression detected in the gills. Its expression could be modulated by LPS, poly(I:C), and infection with Edwardsiella tarda, Aeromonus hydrophila and septicemia viremia of carp virus (SVCV). DrLITAF, when overexpressed, was shown to be located on the cellular membrane and nuclear membrane of HEK293T and ZF4 cells and was associated with the endoplasmic reticulum. Stimulation with LPS resulted in rapid translocation of DrLITAF into the nucleus. In addition, DrLITAF was able to induce cell apoptosis and the expression of caspase 3. The results demonstrate that DrLITAF is involved in the immune defence against bacterial and viral infection and plays a role in regulating inflammation and apoptosis.

Lactobacillus Plantarum 299v Changes miRNA Expression in the Intestines of Piglets and Leads to Downregulation of LITAF by Regulating ssc-miR-450a.

Lactiplantibacillus plantarum subsp. plantarum 299v (L. plantarum 299v) is one of the most important probiotic strains in animal health, but the molecular mechanisms of how it exerts health benefits remain unclear. The purpose of this study was to explore the changes in miRNA expression profiles in the intestinal tissues of piglets by L. plantarum 299v and to explore its possible molecular regulatory mechanism in intestinal function. Neonatal piglets were orally administered L. plantarum 299v daily from 1 to 20days old, and high-throughput sequencing was conducted to analyse the changes in miRNA expression in the jejunum and ileum. The results showed that 370 known porcine miRNAs were identified from eight libraries. Five miRNAs (ssc-miR-21-5p, -143-3p, -194b-5p, -192, and -126-3p) were highly expressed in the intestinal tissues. There were 15 differentially expressed miRNAs between the control group and the L. plantarum group, and only miR-450a was expressed differentially in both intestinal tissues. KEGG analysis revealed that the target genes of the 15 differentially expressed miRNAs were involved in 37 significantly enriched pathways (P < 0.01). Then, quantitative polymerase chain reaction confirmed that the miRNA expression was corresponded well with those from the sequencing. Luciferase reporter assays verified that lipopolysaccharide-induced TNF-α factor is a target of miR-450a. Our results also showed L. plantarum 299v could influence intestinal function by changing the levels of cytokines via miRNA expression. This is the first study to analyse differential expression miRNA profiles in intestinal tissue after L. plantarum 299v treatment and investigate the molecular regulatory mechanism of functional miRNA.

Effects of dietary T-2 toxin on gut health and gut microbiota composition of the juvenile Chinese mitten crab (Eriocheir sinensis)

The current study aims to investigate the effects of dietary T-2 toxin on the intestinal health and microflora in the juvenile Chinese mitten crab (Eriocheir sinensis) with an initial weight 2.00 ± 0.05 g. Juvenile crabs were fed with experimental diets supplemented with T-2 toxin at 0 (control), 0.6 (T1 group), 2.5 (T2 group) and 5.0 (T3 group) mg/kg diet for 8 weeks. Dietary T-2 toxin increased the malondialdehyde (MDA) content and the expression of Kelch-like ECH-associated protein 1 (keap1) gene while the expression of cap ‘n’ collar isoform C (CncC) decreased in the intestine. The activities of glutathione peroxidase (GSH-Px) and total anti-oxidation capacity (T-AOC) in the intestine increased only in the lower dose of dietary T-2. Dietary T-2 toxin significantly increased the mRNA expression of caspase-3, caspase-8, Bax and mitogen-activated protein kinase (MAPK) genes and the ratio of Bax to Bcl-2 accompanied with a reduction of Bcl-2 expression. Furthermore, T-2 toxin decreased the mRNA levels of antimicrobial peptides (AMPs), peritrophic membrane (PM1 and PM2) and immune regulated nuclear transcription factors (Toll-like receptor: TLR, myeloid differentiation primary response gene 88: Myd88, relish and lipopolysaccharide-induced TNF-α factor: LITAF). The richness and diversity of the gut microbiota were also affected by dietary T-2 toxin in T3 group. The similar dominant phyla in the intestine of the Chinese mitten crab in the control and T3 groups were found including Bacteroidetes, Firmicutes, Tenericutes and Proteobacteria. Moreover, the inclusion of dietary T-2 toxin of 4.6 mg/kg significantly decreased the richness of Bacteroidetes and increased the richness of Firmicutes, Tenericutes and Proteobacteria in the intestine. At the genus level, Dysgonomonas and Romboutsia were more abundant in T3 group than those in the control. However, the abundances of Candidatus Bacilloplasma, Chryseobacterium and Streptococcus in T3 group were lower than those in the control. This study indicates that T-2 toxin could cause oxidative damage and immunosuppression, increase apoptosis and disturb composition of microbiota in the intestine of Chinese mitten crab.

Molecular cloning, characterization, and expression analysis of lipopolysaccharide-induced TNF-α factor (LITAF) in the mud crab, Scylla paramamosain

Abstract Lipopolysaccharide-induced TNF-α factor (LITAF) is an important transcription factor mediating the expression of inflammatory cytokines, including TNF-α, which is involved in the innate immune defence against infectious agents. In this study, the Scylla paramamosain LITAF (LITAF) gene was cloned and identified from S. paramamosain using RT-PCR and RACE. The full length cDNA of LITAF was 1116 bp, contained an ORF of 495 bp encoding a polypeptide of 164 amino acids with predicted molecular mass of 17.86 kDa and a theoretical isoelectric point of 8.51. Sequence alignment showed that LITAF possessed a typical LITAF domain with two CXXC motifs and eight conserved cysteine residues. Phylogenetic analysis indicated that LITAF clustered closely with known LITAFs from Eriocheir sinensis and Litopenaeus vannamei. qRT-PCR analysis showed LITAF mRNA transcript expressed ubiquitously in all examined tissues with the highest expression in haemocytes, while lower expression in hepatopancreas. Its expression levels displayed a trend of up-regulation and then returned to its initial value in haemocytes and gill, post challenge with LPS, Poly (I : C), and Vibrio alginolyticus, respectively. Furthermore, the LITAF protein was verified to be located in hyalinocytes, semi-granulocytes and granulocytes, and was distributed throughout the cytoplasm and nucleus, especially in cytoplasm. These results provide a basis for further investigating the roles of LITAF in the immune signaling pathways as a transcription factor in S. paramamosain.

Protective effects of dietary arginine against oxidative damage and hepatopancreas immune responses induced by T-2 toxin in Chinese mitten crab (Eriocheir sinensis)

T-2 toxin is a secondary metabolite produced by Fusarium spp. that is a major cereal and animal feed contaminant. T-2 toxin has numerous adverse effects on animals, including hepatotoxicity. Arginine (Arg) is closely associated with the regulation of immune responses and antioxidant activity in tissues. The objective of the present study was to evaluate the protective effects of dietary Arg against oxidative damage and immune responses of the hepatopancreas induced by T-2 toxin in Chinese mitten crab. According to the results, 3.17% Arg in the diet decreased alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activity in the haemolymph significantly, when compared with the levels of activity in the T-2 toxin group. Arg supplementation also increased superoxide dismutase and glutathione peroxidase activity, while decreasing malondialdehyde concentrations in the hepatopancreas, when compared with the levels in the T-2 toxin group. In addition, 3.17% Arg in the diet increased acid phosphatase and alkaline phosphatase activity in the hepatopancreas, as well as albumin concentrations in the haemolymph, when compared with the T-2 toxin group. Dietary Arg also regulated the expression of antioxidant enzyme-related genes (mitochondrial manganese superoxide dismutase, cytosolic manganese superoxide dismutase, and catalase) and immune related genes (prophenoloxidase, NF-κB-like transcription factor Relish, and lipopolysaccharide-induced TNF-α factor) to alleviate the damage associated with the T-2 toxin. Furthermore, Arg ameliorated damage to the hepatopancreas microstructure in the crabs. The results of the present study indicate that dietary Arg could enhance the antioxidant and immune capacity of Chinese mitten crab against oxidative damage and immune injury to the hepatopancreas induced by T-2 toxin.

Vibrio parahaemolyticus Infection Influenced Trace Element Homeostasis, Impaired Antioxidant Function, and Induced Inflammation Response in Litopenaeus vannamei.

Vibrio parahaemolyticus (V. parahaemolyticus) caused huge diseases and economic losses in shrimp aquaculture. Understanding the infection mechanism might help develop new strategies for controlling pathogen outbreak. Redistribution of trace element homeostasis, accompanied by impairment of antioxidant status and immune response, was observed during various infections. Accordingly, we hypothesized that V. parahaemolyticus infection might influence trace element homeostasis, impair antioxidant function, and induce inflammation response in shrimp. In the present study, the aim of this study was to investigate the influence of V. parahaemolyticus infection on trace element homeostasis, antioxidant status, and inflammation response in Litopenaeus vannamei (L. vannamei). The results showed that compared with the control group, V. parahaemolyticus infection significantly increased (P < 0.05) intestinal V. parahaemolyticus number, serum copper (Cu) concentration at 24, 48, and 72h and significantly increased (P < 0.05) serum zinc (Zn), iron (Fe), and manganese (Mn) concentrations at 24h but decreased (P < 0.05) at 72h. The intestinal gene expressions of metal transporters ZIP13, CTR1, and MT1 were significantly decreased at 24, 48, and 72h, and DMT1 was significantly decreased at 48h and 72h in the infection group. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were suppressed at 48h and 72h, and the malondialdehyde (MDA) content was increased at 24, 48, and 72h in the infection group; the pro-inflammatory genes including necrosis factor-α (TNF-α), lipopolysaccharide-induced TNF-α factor (LITAF), and Ras-related protein Rab6A (RAB6A) were significantly upregulated at 48 and 72h in the infection group. These results suggest that V. parahaemolyticus infection influenced trace element homeostasis, impaired antioxidant function, and induced inflammation response in L. vannamei, which might help understand the infection mechanism. The results provide a better understanding of the L. vannamei and V. parahaemolyticus interactions and may deliver the basis for further research in preventing the bacterial diseases.

Novel EGR2 variant that associates with Charcot-Marie-Tooth disease when combined with lipopolysaccharide-induced TNF-α factor T49M polymorphism.

ObjectiveTo identify novel genetic mechanisms causing Charcot-Marie-Tooth (CMT) disease.MethodsWe performed a next-generation sequencing study of 34 genes associated with CMT in a patient with peripheral neuropathy.ResultsWe found a non–previously described mutation in EGR2 (p.P397H). P397H mutation is located within the loop that connects zinc fingers 2 and 3, a pivotal domain for the activity of this transcription factor. Using promoter activity luciferase assays, we found that this mutation promotes decreased transcriptional activity of EGR2. In this patient, we also found a previously described nonpathogenic polymorphism in lipopolysaccharide-induced TNF-α factor (LITAF) (p.T49M). We show that the p.T49M mutation decreases the steady-state levels of the LITAF protein in Schwann cells. Loss of function of LITAF has been shown to produce deregulation in the NRG1-erbB signaling, a pivotal pathway for EGR2 expression by Schwann cells. Surprisingly, our segregation study demonstrates that p.P397H mutation in EGR2 is not sufficient to produce CMT disease. Most notably, only those patients expressing simultaneously the LITAF T49M polymorphism develop peripheral neuropathy.ConclusionsOur data support that the LITAF loss-of-function interferes with the expression of the transcriptional-deficient EGR2 P397H mutant hampering Schwann cell differentiation and suggest that in vivo both genes act in tandem to allow the proper development of myelin.

Improvement of dietary N-acetylcysteine on growth inhibition and intestinal damage induced by β-conglycinin in juvenile Chinese mitten crabs (Eriocheir sinensis)

β-conglycinin is a major component of feed allergens that can cause inflammation and dysfunction of intestinal digestion and absorption in fish, but little is known in crab. This study investigated the mitigative effects of N-acetylcysteine (NAC) on growth inhibition and intestinal damage induced by β-conglycinin in juvenile Chinese mitten crabs (Eriocheir sinensis). All three experimental diets contained 70 g/kg β-conglycinin, but with three levels of NAC in each diet (0, 0.5 and 1 g/kg respectively). The control diet did not contain β-conglycinin nor NAC, but all four diets were isonitrogenous and isolipidic. Juvenile crab (3.06 ± 0.03 g) were respectively fed with these diets for eight weeks. Dietary inclusion of 1 g/kg NAC significantly improved crab survival and weight gain than those of the negative control, and showed no difference with the control group. The crabs fed diets containing β-conglycinin had lower glutathione and catalase activities but higher malondialdehyde in the intestine than those in the control group. Moreover, dietary β-conglycinin improved the level of histamine in the intestine and increased the activity of diamine oxidase in serum. The activities of lipase in hepatopancreas and trypsin in both hepatopancreas and intestine were restrained by β-conglycinin, while addition of NAC reversed these negative effects. The addition of NAC restored the impaired immunity and morphological structure of the intestine. Moreover, dietary NAC downregulated mRNA levels of pro-inflammatory factor genes (lipopolysaccharide-induced TNF-α factor, EsRelish and interleukin-2 enhancer-binding factor 2) while upregulated the structural integrity peritrophic membrane related gene expression levels (peritrophin-like gene, peritrophic 1 and 2). This study indicates that N-acetylcysteine is a useful feed additive to ameliorate the growth inhibition, intestinal oxidative damage and inflammation induced by β-conglycinin in crab diets.

Effects of dietary supplementation with icariin on growth performance, antioxidant capacity and non-specific immunity of Chinese mitten crab (Eriocheir sinensis)

We investigated the effects of icariin (ICA) on growth performance, antioxidant capacity and non-specific immunity in Chinese mitten crab (Eriocheir sinensis). A total of 200 healthy crabs (average weight: 33.58 ± 0.05 g) were randomly assigned to four treatments with five replicates, each with ten individuals per pool. There were four dietary treatments: the control group (fed with the basal diet), the ICA 50 group, the ICA100 group, and the ICA 200 group (fed with the basal diet supplemented with 50, 100, and 200 mg/kg ICA, respectively). These diets were provided for 8 weeks. Results indicated that ICA100 crabs had higher weight gain (WG), specific growth rate (SGR) and survival rate (SR) than the controls. Protein carbonyl content (PCC) and malondialdehyde (MDA) concentrations in the haemolymph and hepatopancreas of ICA100 crabs were significantly lower than in the control group, while the superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were significantly higher. The activities of PO, LZM, ACP and AKP were significantly enhanced with ICA supplementation at 50 and 100 mg/kg, yet decreased subsequently at 200 mg/kg. Furthermore, supplementation of 100 mg/kg ICA up-regulated the mRNA expression of prophenoloxidase (proPO), catalase (CAT), mitochondrial manganese superoxide dismutase (mtMnSOD), thioredoxin-1 (Trx1) and peroxiredoxin 6 (Prx6), while the mRNA expression of toll like receptors (TLRs), NF-κB-like transcription factor Relish and lipopolysaccharide-induced TNF-α factor (LITAF) were down-regulated in the hepatopancreas (P < 0.05). These findings indicate that dietary ICA supplementation at an optimum dose of 100 mg/kg may be effective in improving growth performance, antioxidant capability and non-specific immunity of Chinese mitten crab.

Molecular cloning and characterization of grouper Krϋppel-like factor 9 gene: Involvement in the fish immune response to viral infection

Krϋppel-like factor 9 (KLF9) is a member of the SP/KL family, which are transcription factors implicated in several biological processes, including cell proliferation, differentiation, development and apoptosis. Studies have focused on the function of KLF9 in mammalian disease and the immune system, such as its regulatory role in the growth of tumors and its impact on interferon-related genes and inflammatory cytokines. In fish, little is known about the role of KLF9, especially its regulatory function in the innate antiviral immune response. In this study, we characterized the grouper KLF9 gene (EcKLF9) and investigated its role in viral infection. Amino acid alignment analysis showed that EcKLF9 was approximately 228 amino acids long and contained a typical three-tandem Krϋppel-like zinc fingers. Phylogenetic tree analysis revealed that EcKLF9 clustered with three fish species: Amphiprion ocellaris, Acanthochromis pollyacanthus and Stegastes partitus. Comparison analyses showed that the three Kruppel-like zinc finger domains of KLF9 were highly conserved in different fish species. Tissue expression analysis showed that EcKLF9 was constitutively expressed in all 12 tissues tested, in the healthy grouper, the highest expression being detected in the gonads. The relative expression levels of EcKLF9 in the head kidney, spleen and brain was significantly increased during red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV) infections. Using fluorescence microscopy, EcKLF9 was primarily localized to the nucleus and cytoplasm. The in vitro ectopic expression of EcKLF9 significantly increased the severity of vacuoles induced by RGNNV and the cytopathic effect progression evoked by SGIV infection. Real-time PCR results showed that the transcription levels of viral genes, such as the Singapore grouper iridovirus infection genes, MCP (major capsid protein), LITAF (lipopolysaccharide induced TNF-α factor), VP19 (envelop protein) ICP-18 (infected cell protein-18) and the red-spotted grouper nervous necrosis virus genes, CP (coat protein), RdRp (RNA-dependent RNA polymerase), were all significantly increased in EcKLF9 overexpressing cells, when compared to control cells. Furthermore, western blotting analyses showed that protein levels of the RGNNV gene, CP and the SGIV gene, MCP were also increased in EcKLF9 overexpressing cells, suggesting EcKLF9 may promote viral activity against iridovirus and nodavirus, in vitro. Moreover, the overexpression of EcKLF9 significantly inhibited the expression of several interferon related cytokines and several inflammatory cytokines. Accordingly, we speculate that EcKLF9 may exert stimulatory effects on RGNNV and SGIV replication, through the negative regulation of host immune and inflammation responses.

Effects of glycinin and β-conglycinin on growth performance and intestinal health in juvenile Chinese mitten crabs (Eriocheir sinensis)

This study investigates the effects of two soybean antigens (glycinin and β-conglycinin) as an antinutritional substance in the diet on the growth, digestive ability, intestinal health and microbiota of juvenile Chinese mitten crabs (Eriocheir sinensis). The isonitrogenous and isolipidic diets contained two soybean antigens at two levels each (70 and 140 g/kg β-conglycinin, 80 and 160 g/kg glycinin) and a control diet without β-conglycinin or glycinin supplementation, and were used respectively to feed juvenile E. sinensis for seven weeks. Dietary inclusion of either glycinin or β-conglycinin significantly reduced crab survival and weight gain. The crabs fed diets containing soybean antigens had higher malondialdehyde concentrations and lower catalase activities in the intestine than those in the control. The activities of trypsin and amylase in the intestine were suppressed by dietary β-conglycinin and glycinin. Dietary glycinin or β-conglycinin impaired the immunity and morphological structure of intestine, especially the peritrophic membrane. The mRNA expression of constitutive and inducible immune responsive genes (lipopolysaccharide-induced TNF-α factor and interleukin-2 enhancer-binding factor 2) increased while the mRNA expression of the main genes related to the structural integrity peritrophic membrane (peritrophin-like gene and peritrophic 2) significantly decreased in the groups with soybean antigen addition. Soybean antigen could also change the intestinal microbial community. The abundance of pathogenic bacteria (Ochrobactrum, Burkholderia and Pseudomonas) increased significantly in both soybean antigen groups. Although pathogenic bacteria Vibrio were up-regulated in the glycinin group, the abundance of Dysgonomonas that degraded lignocellulose and ameliorated the gut environment decreased in the glycinin group. This study indicates that existence of soybean antigens (glycinin or β-conglycinin) could induce gut inflammation, reshape the community of gut microbiota, and cause digestive dysfunction, ultimately leading to impaired growth in crabs.

Dietary fructooligosaccharide can mitigate the negative effects of immunity on Chinese mitten crab fed a high level of plant protein diet

An 8-week feeding trial was carried out under controlled condition to evaluate the effect of dietary fructooligosaccharide (FOS) on growth performance, whole body composition, antioxidant status and immunity of crabs fed high levels of plant protein diets. Thus, six experimental diets were formulated (designated as F0P50, F0P60, F0P70, F0.2P50, F0.2P60 and F0.2P70), which contain two FOS levels (0 or 0.2%) and three plant protein levels (50, 60, or 70%) according to a 2 × 3 factorial design. The results showed that weight gain increased significantly as dietary plant protein level decreased from 70% to 50%. At 50% plant protein level, the addition of 0.2% FOS can significantly elevate weight gain (WG) (P < 0.05). The highest value in survival rate was observed in crabs fed F0.2P50 and F0.2P60 diet. Crabs fed F0.2P50 diet showed significantly higher crude protein content (P < 0.05) compared with those in other groups, but there were no significant differences in the contents of moisture, crude lipid and ash among all groups (P > 0.05). Catalase (CAT) activity in crabs fed F0.2P50 increased significantly (P < 0.05) compared with crabs fed F0P60, F0P70, F0.2P60 and F0.2P70, but malondialdehyde (MDA) concentrations decreased significantly (P < 0.05). Meanwhile, nitric oxide (NO) concentration, acid phosphatase (ACP) and alkaline phosphatase (AKP) activities of crabs fed 0.2% FOS diets increased significantly (P < 0.05) compared with crabs fed 0% FOS diets. The expressions of prophenoloxidase (propo) was significantly (P < 0.05) affected only by dietary plant protein levels with the highest values observed in 50% plant protein diet, whereas the opposite was true for Myeloid differentiation factor 88 (myd88). The mRNA expressions of mitochondrial manganese superoxide dismutase (mtmnsod), lipopolysaccharide-induced TNF-α factor (litaf) and toll like receptors (tlrs) were significantly affected (P < 0.05) by both FOS and plant protein levels. The cytosolic manganese superoxide dismutase (cytmnsod) mRNA expressions in F0.2P50 and F0.2P60 groups were significantly higher than those in F0P70 and F0.2P70 groups. The results in this study indicated that supplementation with 0.2% FOS can enhance growth performance in crabs fed lower plant protein diets and as well improve immunity in those fed with higher plant protein diets.

Effects of dietary fructooligosaccharide on the growth, antioxidants, immunity and disease resistance of Chinese mitten crab

This study aimed to investigate the effects of dietary fructooligosaccharide (FOS) on the growth, antioxidant capability, immune response and disease resistance of Chinese mitten crab Eriocheir sinensis. Groups of crab (17.74±0.39g) were distributed into 18 cement pools (33 crabs per pool) with replicates and fed experimental diets supplemented with FOS at 0, 0.5, 1.0, 1.5, 2.0 and 2.5g/kg (designated as control, TF1, TF2, TF3, TF4 and TF5, respectively). After 60days of feeding trial, crabs were challenged by Aeromonas hydrophila and the survival was recorded for the next 96h. The results indicated that crabs fed TF4 had significantly higher weight gain (WG) than crabs fed control diet, whereas WG decreased with FOS level increasing to 2.5g/kg. There was no significant difference (P>0.05) in SGR among all experimental groups. Catalase (CAT) and superoxide dismutase (SOD) activities in crabs fed TF4 diet were significantly higher than those in control group, while malondialdehyde (MDA) concentrations in crabs fed TF4 diet were significantly lower than that in control group. Crabs fed TF4 diet showed significantly higher alkaline phosphatase (AKP) activity compared to the control group. There was a trend that acid phosphatase (ACP) activity was elevated with FOS levels increasing up to 2.0g/kg and decreased subsequently, and crabs fed TF4 diet showed significantly higher ACP activity compared to the control group. Supplementation of 2.0g/kg FOS up-regulated the mRNA expression of interleukin-2 enhancer binding factor 2 (ILF2) and peroxinectin (PX), and down-regulated the mRNA expression of lipopolysaccharide-induced TNF-α factor (LITAF). Bifidobacteriales abundance in the digestive tract increased significantly with dietary FOS levels increasing from 1.5 to 2.5g/kg. Crabs fed TF4 showed significantly higher Bacteroides abundance compare to the other groups. After Aeromonas hydrophila challenge, the highest relative percent survival (25.00%) was observed in crabs fed TF4 diet, which was significantly higher than that in control group. Taken together, 2.0g/kg FOS supplementation might be effective in improving growth performance, antioxidant capability, immune response and disease resistance of Chinese mitten crab.

Abstract 830: MiRNA-106a and LITAF are novel modulators of prostate cancer radioresistance

Abstract Prostate cancer (PCa) is the second most prevalent cancer affecting men worldwide, with radiotherapy being a primary treatment modality. PCa recurrence is a major clinical problem with up to a 40% biochemical recurrence rate at five years after external beam radiotherapy (ionizing radiation, IR). Therefore, there is an unmet need to characterize radioresistance in order to improve therapy and patient outcomes. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. MiRNAs are aberrantly expressed in cancer, specifically, miR-106a has been found to be overexpressed in various cancer types. The purpose of this study is to determine whether miR-106a confers IR resistance and tumor aggression in PCa. Utilizing The Cancer Genome Atlas Data Portal, bioinformatics analysis discovered that miR-106a was significantly overexpressed in PCa samples relative to normal prostate. This suggests that miR-106a is involved in prostate carcinogenesis. Clonogenic survival assays revealed that cells overexpressing miR-106a had increased survival after IR treatment compared to control cells. Proliferation assays showed that miR-106a cells had a higher proliferation rate than control cells in both unirradiated and IR-treated cells. Affymetrix gene array analysis was used to identify possible targets of miR-106a. Combining results from the gene array, in silico prediction algorithms, and in vitro analyses, we identified lipopolysaccharide-induced TNFα factor (LITAF) as a target of miR-106a. In addition, LITAF knockdown lead to increased proliferation and clonogenic survival following IR, recapitulating the phenotype of miR-106a overexpression. Cells were stained for β-galactosidase expression following IR to assess senescence, as a mode of cell death. We found that both miR-106a overexpression and LITAF knockdown resulted in significantly fewer senescent cells post-IR. Upon examination of critical DNA damage response genes, we found that miR-106a overexpression and LITAF knockdown increased expression of ATM mRNA and protein. Upregulation of ATM is associated with IR resistance, elucidating the mechanism by which miR-106a overexpression and LITAF knockdown are involved in radioresistance. Tumor xenograft experiments, using a PCa cell line stably overexpressing miR-106a, confirmed that miR-106a increases proliferation compared to control tumors before and after IR. Thus, our results strongly suggest that miR-106a is involved in PCa aggression and confers a radiation-resistant phenotype, by targeting the novel radiation response gene LITAF. Note: This abstract was not presented at the meeting. Citation Format: Christianne Hoey, Jessica Ray, Paul Boutros, Stanley K. Liu. MiRNA-106a and LITAF are novel modulators of prostate cancer radioresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 830. doi:10.1158/1538-7445.AM2017-830

Cgi-miR-92d indirectly regulates TNF expression by targeting CDS region of lipopolysaccharide-induced TNF-α factor 3 (CgLITAF3) in oyster Crassostrea gigas.

Tumor necrosis factor alpha (TNF-α) mediated inflammatory response plays indispensable roles in organisms defending against the invaded bacteria, during which microRNAs have been found crucial by controlling multiple TNF-α-related genes. In the present study, cgi-miR-92d was annotated as a member of miR-17-92 family and could target the CDS region of lipopolysaccharide (LPS)-induced TNF-α factor (CgLITAF3) in oyster Crassostrea gigas. It was observed that cgi-miR-92d could be vigorously modulated by Vibrio splendidus or LPS stimulation while CgLITAF3 altered oppositely. Two putative binding sites of cgi-miR-92d were then found at CDS region of CgLITAF3. The interaction between cgi-miR-92d and CgLITAF3 was subsequently verified both invitro and invivo. As a result, a significant decrease of cellular luminescence was observed in CgLITAF3 luciferase reporter assay when cgi-miR-92d was overexpressed. The luminescent decrease was then recuperated when cgi-miR-92d inhibitor was co-transfected with miRNA mimics. Besides, CgLITAF3 transcripts were significantly down-regulated when cgi-miR-92d was overexpressed invivo during V. splendidus challenge. Gain-of-function assay of CgLITAF3 was then conducted in HEK293T cells to verify its function. Consequently, a significant increase of TNF-α was observed during the assay. At the meantime, CgTNF was also down-regulated in gain-of-function assay of cgi-miR-92 invivo, which was a member of TNF superfamily in oysters which could be robustly induced after pathogen stimulation. Together, these results verify the interaction between CgLITAF3 and cgi-miR-92d, which might dedicate crucially in the repaid activation of CgTNF expression during inflammatory response of oysters.

Tiger frog virus ORF080L protein interacts with LITAF and impairs EGF-induced EGFR degradation

Tiger frog virus (TFV) belongs to the genus Ranavirus, family Iridoviridae, and causes severe mortality in commercial cultures in China. TFV ORF080L is a gene homolog of lipopolysaccharide-induced TNF-α factor (LITAF), which is a regulator in endosome-to-lysosome trafficking through its function in the endosomal sorting complex required for transport machinery. The characteristics and biological roles of TFV ORF080L were identified. TFV ORF080L was predicted to encode an 84-amino acid peptide (VP080L). It had high-sequence identity with mammalian LITAF, but lacked the N-terminus of LITAF, which contains two PPXY motifs. Transcription and protein level analyses showed that TFV ORF080L was a late viral gene. Localization in the virons also showed that TFV VP080L was a viral structural protein. Immunofluorescence staining showed that TFV ORF080L was predominantly colocalized with plasma membrane and partly distributed with the late endosome in infected HepG2 cells. SiRNA-mediated TFV ORF080L silencing decreased viral reproduction. Moreover, TFV ORF080L interacted with human/zebrafish LITAF and impaired EGF-induced EGFR degradation, thereby indicating that TFV ORF080L played a role in endosome-to-lysosome trafficking. These findings suggested that TFV ORF080L might negate the function of cellular LITAF to impair endosomal sorting and trafficking. Results provide a clue to the link between the dysregulated endosomal trafficking and iridovirus pathogenesis.

Using In Vitro Immunomodulatory Properties of Lactic Acid Bacteria for Selection of Probiotics against Salmonella Infection in Broiler Chicks.

Poultry is known to be a major reservoir of Salmonella. The use of lactic acid bacteria has become one of successful strategies to control Salmonella in poultry. The purpose of this study was to select lactic acid bacteria strains by their in vitro immunomodulatory properties for potential use as probiotics against Salmonella infection in broiler chicks. Among 101 isolated lactic acid bacteria strains, 13 strains effectively survived under acidic (pH 2.5) and bile salt (ranging from 0.1% to 1.0%) conditions, effectively inhibited growth of 6 pathogens, and adhered to Caco-2 cells. However, their in vitro immunomodulatory activities differed significantly. Finally, three strains with higher in vitro immunomodulatory properties (Lactobacillus plantarum PZ01, Lactobacillus salivarius JM32 and Pediococcus acidilactici JH231) and three strains with lower in vitro immunomodulatory activities (Enterococcus faecium JS11, Lactobacillus salivarius JK22 and Lactobacillus salivarius JM2A1) were compared for their inhibitory effects on Salmonella adhesion and invasion to Caco-2 cells in vitro and their antimicrobial effects in vivo. The former three strains inhibited Salmonella adhesion and invasion to Caco-2 cells in vitro, reduced the number of Salmonella in intestinal content, spleen and liver, reduced the levels of lipopolysaccharide-induced TNF-α factor (LITAF), IL-1β, IL-6 and IL-12 in serum and increased the level of IL-10 in serum during a challenge study in vivo more efficiently than the latter three strains. These results suggest that in vitro immunomodulatory activities could be used as additional parameters to select more effective probiotics as feed supplements for poultry.