Isolation of Naegleria lustrarea n. sp. (Excavata, Discoba, Heterolobosea) from the feces of Ambystoma annulatum (Ringed Salamander) in Northwest Arkansas.

The salamander, Ambystoma annulatum, is considered a "species of special concern" in the state of Arkansas, USA, due to its limited geographic range, specialized habitat requirements and low population size. Although metazoan parasites have been documented in this salamander species, neither its native protists nor microbiome have yet been evaluated. This is likely due to the elusive nature and under-sampling of the animal. Here, we initiate the cataloguing of microbial associates with the identification of a new heterlobosean species, Naegleria lustrarea n. sp. (Excavata, Discoba, Heterolobosea), isolated from feces of an adult A. annulatum.

FLA11 and FLA12 glycoproteins fine-tune stem secondary wall properties in response to mechanical stresses.

Secondary cell walls (SCWs) in stem xylem vessel and fibre cells enable plants to withstand the enormous compressive forces associated with upright growth. It remains unclear if xylem vessel and fibre cells can directly sense mechanical stimuli and modify their SCW during development. We provide evidence that Arabidopsis SCW-specific Fasciclin-Like Arabinogalactan-proteins 11 (FLA11) and 12 (FLA12) are possible cell surface sensors regulating SCW development in response to mechanical stimuli. Plants overexpressing FLA11 (OE-FLA11) showed earlier SCW development compared to the wild-type (WT) and altered SCW properties that phenocopy WT plants under compression stress. By contrast, OE-FLA12 stems showed higher cellulose content compared to WT plants, similar to plants experiencing tensile stress. fla11, OE-FLA11, fla12, and OE-FLA12 plants showed altered SCW responses to mechanical stress compared to the WT. Quantitative polymerase chain reaction (qPCR) and RNA-seq analysis revealed the up-regulation of genes and pathways involved in stress responses and SCW synthesis and regulation. Analysis of OE-FLA11 nst1 nst3 plants suggests that FLA11 regulation of SCWs is reliant on classical transcriptional networks. Our data support the involvement of FLA11 and FLA12 in SCW sensing complexes to fine-tune both the initiation of SCW development and the balance of lignin and cellulose synthesis/deposition in SCWs during development and in response to mechanical stimuli.

Target identification of anti-diabetic and anti-obesity flavonoid derivative (Fla-CN).

Fla-CN is a flavonoid derivative with anti-diabetic and anti-obesity effects; however, its biological targets are still unknown. In this study, we developed bifunctional affinity-based probes to identify the direct targets of Fla-CN. When using probe 3, we observed the co-location of probe 3 and mitochondria in both HepG2 and 3T3-L1 cells. The putative target proteomes were obtained using activity-based protein profiling (ABPP) and photo-affinity labelling. Pyruvate carboxylase, mitochondrial malate dehydrogenase, mitochondrial complex I, and F1FO-ATPase were validated as the direct targets of Fla-CN by surface plasmon resonance (SPR) and biochemical assays. It was elucidated that the Tyr651, Gln870 and Lys912 were the key amino acid residues near the binding site of pyruvate carboxylase with Fla-CN. The direct interaction of Fla-CN and the above four targets allowed elucidation of its complicated molecular mechanism, including the activation of adenosine 5-monophosphate (AMP)-activated protein kinase (AMPK), and the inhibition of gluconeogenesis. Further investigation for activation of AMPK in normal and insulin resistance (IR) HepG2 cells, indicated that Fla-CN could target insulin resistance tissues.

Construction of a mutant Bacillus subtilis strain for high purity poly-γ-glutamic acid production.

OBJECTIVE: To construct a Bacillus subtilis strain for improved purity of poly-γ-glutamic acid. RESULTS: The construction of strain GH16 was achieved by knocking out five genes encoding extracellular proteins and an operon from Bacillus subtilis G423. We then analyzed the amount of protein impurities in the γ-PGA produced by the resulting strain GH16/pHPG, which decreased from 1.48 to 1.39%. Subsequently the fla-che operon, PBSX, as well as the yrpD, ywoF and yclQ genes were knocked out successively, resulting in the mutant strains GH17, GH18 and GH19. Ultimately, the amount of protein impurities was reduced from 1.48 to 0.83%. In addition, the amount of polysaccharide impurities in the γ-PGA was also decreased from 2.21 to 1.93% after knocking out the epsA-O operon. CONCLUSIONS: The high purity γ-PGA producer was constructed, and the resulting strain was a promising platform for the manufacture of other highly pure extracellular products and secretory proteins.

Comprehensive Analysis of Arabinogalactan Protein-Encoding Genes Reveals the Involvement of Three BrFLA Genes in Pollen Germination in Brassica rapa.

Arabinogalactan proteins (AGPs) are a superfamily of hydroxyproline-rich glycoproteins that are massively glycosylated, widely implicated in plant growth and development. No comprehensive analysis of the AGP gene family has been performed in Chinese cabbage (Brassica rapa ssp. chinensis). Here, we identified a total of 293 putative AGP-encoding genes in B. rapa, including 25 classical AGPs, three lysine-rich AGPs, 30 AG-peptides, 36 fasciclin-like AGPs (FLAs), 59 phytocyanin-like AGPs, 33 xylogen-like AGPs, 102 other chimeric AGPs, two non-classical AGPs and three AGP/extensin hybrids. Their protein structures, phylogenetic relationships, chromosomal location and gene duplication status were comprehensively analyzed. Based on RNA sequencing data, we found that 73 AGP genes were differentially expressed in the floral buds of the sterile and fertile plants at least at one developmental stage in B. rapa, suggesting a potential role of AGPs in male reproductive development. We further characterized BrFLA2, BrFLA28 and BrFLA32, three FLA members especially expressed in anthers, pollen grains and pollen tubes. BrFLA2, BrFLA28 and BrFLA32 are indispensable for the proper timing of pollen germination under high relative humidity. Our study greatly extends the repertoire of AGPs in B. rapa and reveals a role for three members of the FLA subfamily in pollen germination.

Cross-Talk of Toll-Like Receptor 5 and Mu-Opioid Receptor Attenuates Chronic Constriction Injury-Induced Mechanical Hyperalgesia through a Protein Kinase C Alpha-Dependent Signaling.

Recently, Toll-like receptors (TLRs), a family of pattern recognition receptors, are reported as potential modulators for neuropathic pain; however, the desired mechanism is still unexplained. Here, we operated on the sciatic nerve to establish a pre-clinical rodent model of chronic constriction injury (CCI) in Sprague-Dawley rats, which were assigned into CCI and Decompression groups randomly. In Decompression group, the rats were performed with nerve decompression at post-operative week 4. Mechanical hyperalgesia and mechanical allodynia were obviously attenuated after a month. Toll-like receptor 5 (TLR5)-immunoreactive (ir) expression increased in dorsal horn, particularly in the inner part of lamina II. Additionally, substance P (SP) and isolectin B4 (IB4)-ir expressions, rather than calcitonin-gene-related peptide (CGRP)-ir expression, increased in their distinct laminae. Double immunofluorescence proved that increased TLR5-ir expression was co-expressed mainly with IB4-ir expression. Through an intrathecal administration with FLA-ST Ultrapure (a TLR5 agonist, purified flagellin from Salmonella Typhimurium, only the CCI-induced mechanical hyperalgesia was attenuated dose-dependently. Moreover, we confirmed that mu-opioid receptor (MOR) and phospho-protein kinase Cα (pPKCα)-ir expressions but not phospho-protein kinase A RII (pPKA RII)-ir expression, increased in lamina II, where they mostly co-expressed with IB4-ir expression. Go 6976, a potent protein kinase C inhibitor, effectively reversed the FLA-ST Ultrapure- or DAMGO-mediated attenuated trend towards mechanical hyperalgesia by an intrathecal administration in CCI rats. In summary, our current findings suggest that nerve decompression improves CCI-induced mechanical hyperalgesia that might be through the cross-talk of TLR5 and MOR in a PKCα-dependent manner, which opens a novel opportunity for the development of analgesic therapeutics in neuropathic pain.

The Role of First Language Attrition in Persian Idiomatic Expressions.

In recent years, despite the fact that many researchers have devoted much of their attention to second language attrition, not much focus has been given to first language attrition (FLA) specifically among Iranian immigrants. The present study attempts to describe FLA in the semantic domain of idiomatic expression and effect of length of residence among Persian native speakers who live in Iran as well as those who migrate to English-speaking countries. The present study explores language attrition in three migrant populations (Persians in the United States, Australia, and Canada). The participants were selected through convenience sampling. Furthermore, to find the impact of length of residence, the immigrants were divided into two groups comprising short- and long-term residence groups. The instrument applied by the researchers for data collection included a researcher-devised idiomatic expression test to assess immigrants' level of idiom comprehension and demographic information questionnaire to have a better understanding of immigrants' background characteristics. Results revealed that the immigrants underwent FLA and the rate of attrition was higher in long-term immigrants. The results are in harmony with the Activation Threshold Hypothesis showing the language attrition among fewer L1 users. The finding of this study sheds new light on the understanding of the concept of first language attrition in migration studies.

Evolution Analysis of the Fasciclin-Like Arabinogalactan Proteins in Plants Shows Variable Fasciclin-AGP Domain Constitutions.

The fasciclin-like arabinogalactan proteins (FLAs) play important roles in plant development and adaptation to the environment. FLAs contain both fasciclin domains and arabinogalactan protein (AGP) regions, which have been identified in several plants. The evolutionary history of this gene family in plants is still undiscovered. In this study, we identified the FLA gene family in 13 plant species covering major lineages of plants using bioinformatics methods. A total of 246 FLA genes are identified with gene copy numbers ranging from one (Chondrus crispus) to 49 (Populus trichocarpa). These FLAs are classified into seven groups, mainly based on the phylogenetic analysis of plant FLAs. All FLAs in land plants contain one or two fasciclin domains, while in algae, several FLAs contain four or six fasciclin domains. It has been proposed that there was a divergence event, represented by the reduced number of fasciclin domains from algae to land plants in evolutionary history. Furthermore, introns in FLA genes are lost during plant evolution, especially from green algae to land plants. Moreover, it is found that gene duplication events, including segmental and tandem duplications are essential for the expansion of FLA gene families. The duplicated gene pairs in FLA gene family mainly evolve under purifying selection. Our findings give insight into the origin and expansion of the FLA gene family and help us understand their functions during the process of evolution.

One-year survey of opportunistic premise plumbing pathogens and free-living amoebae in the tap-water of one northern city of China.

In this study, qPCR was used to quantify opportunistic premise plumbing pathogens (OPPPs) and free-living amoebae in 11 tap water samples collected over four seasons from a city in northern China. Results demonstrated that the average numbers of gene copies of Legionella spp. and Mycobacterium spp. were significantly higher than those of Aeromonas spp. (p < 0.05). Legionella spp. and Mycobacterium spp. were 100% (44/44) positively detected while P. aeruginosa and Aeromonas spp. were 79.54% (35/44) and 77.27% (34/44) positively detected. Legionella pneumophila was only detected in 4 samples (4/44), demonstrating its occasional occurrence. No Mycobacterium avium or Naegleria fowleri was detected in any of the samples. The average gene copy numbers of target OPPPs were the highest in summer, suggesting seasonal prevalence of OPPPs. Average gene copy numbers of OPPPs in the taps of low-use-frequency were higher than in taps of high-use-frequency, but the difference was not significant for some OPPPs (p > 0.05). Moderate negative correlations between the chlorine concentration and the gene copy numbers of OPPPs were observed by Spearman analysis (rs ranged from -0.311 to -0.710, p < 0.05). However, no significant correlations existed between OPPPs and AOC, BDOC, or turbidity. Moderate positive correlations were observed between the target microorganisms, especially for Acanthamoeba spp., through Spearman analysis (p < 0.05). Based on our studies, it is proposed that disinfectant concentration, season, taps with different-use frequency, OPPP species, and potential microbial correlations should be considered for control of OPPPs in tap water.

Reduced expression of selected FASCICLIN-LIKE ARABINOGALACTAN PROTEIN genes associates with the abortion of kernels in field crops of Zea mays (maize) and of Arabidopsis seeds.

Abortion of fertilized ovaries at the tip of the ear can generate significant yield losses in maize crops. To investigate the mechanisms involved in this process, 2 maize hybrids were grown in field crops at 2 sowing densities and under 3 irrigation regimes (well-watered control, drought before pollination, and drought during pollination), in all possible combinations. Samples of ear tips were taken 2-6 days after synchronous hand pollination and used for the analysis of gene expression and sugars. Glucose and fructose levels increased in kernels with high abortion risk. Several FASCICLIN-LIKE ARABINOGALACTAN PROTEIN (FLA) genes showed negative correlation with abortion. The expression of ZmFLA7 responded to drought only at the tip of the ear. The abundance of arabinogalactan protein (AGP) glycan epitopes decreased with drought and pharmacological treatments that reduce AGP activity enhanced the abortion of fertilized ovaries. Drought also reduced the expression of AthFLA9 in the siliques of Arabidopsis thaliana. Gain- and loss-of-function mutants of Arabidopsis showed a negative correlation between AthFLA9 and seed abortion. On the basis of gene expression patterns, pharmacological, and genetic evidence, we propose that stress-induced reductions in the expression of selected FLA genes enhance abortion of fertilized ovaries in maize and Arabidopsis.

Optimal pcr primers for rapid and accurate detection of Aspergillus flavus isolates.

Aspergillus flavus is among the most devastating opportunistic pathogens of several food crops including rice, due to its high production of carcinogenic aflatoxins. The presence of these organisms in economically important rice strip farming is a serious food safety concern. Several polymerase chain reaction (PCR) primers have been designed to detect this species; however, a comparative assessment of their accuracy has not been conducted. This study aims to identify the optimal diagnostic PCR primers for the identification of A. flavus, among widely available primers. We isolated 122 A. flavus native isolates from randomly collected rice strips (N = 300). We identified 109 isolates to the genus level using universal fungal PCR primer pairs. Nine pairs of primers were examined for their PCR diagnostic specificity on the 109 isolates. FLA PCR was found to be the optimal PCR primer pair for specific identification of the native isolates, over aflP(1), aflM, aflA, aflD, aflP(3), aflP(2), and aflR. The PEP primer pair was found to be the most unsuitable for A. flavus identification. In conclusion, the present study indicates the powerful specificity of the FLA PCR primer over other commonly available diagnostic primers for accurate, rapid, and large-scale identification of A. flavus native isolates. This study provides the first simple, practical comparative guide to PCR-based screening of A. flavus infection in rice strips.

Phylogenetic distribution of the euryarchaeal archaellum regulator EarA and complementation of a Methanococcus maripaludis ∆earA mutant with heterologous earA homologues.

Archaella are the swimming organelles in the Archaea. Recently, the first archaellum regulator in the Euryarchaeota, EarAMma, was identified in Methanococcus maripaludis, one of the model organisms used for archaellum studies. EarAMma binds to 6 bp consensus sequences upstream of the fla promoter to activate the transcription of the fla operon, which encodes most of the proteins required for archaella synthesis. In this study, synteny analysis showed that earA homologues are widely distributed in the phylum of Euryarchaeota, with the notable exception of extreme halophiles. We classified Euryarchaeota species containing earA homologues into five classes based on the genomic location of the earA genes relative to fla and chemotaxis operons. EarA homologues from Methanococcus vannielii, Methanothermococcus thermolithotrophicus and Methanocaldococcus jannaschii successfully complemented the function of EarAMma in a ΔearAMma mutant, demonstrated by the restoration of FlaB2 expression in Western blot analysis and the appearance of archaella on the cell surface in complemented cells. Furthermore, the 6 bp consensus sequence was also found in the fla promoter region in these methanogens, indicating that the EarA homologues ly use a similar mechanism to activate transcription of the fla operons in their own hosts. Attempts to demonstrate complementation of the function of EarAMma in a ΔearAMma mutant by the EarA homologue of Pyrococcus furiosus were unsuccessful, despite the presence of a copy of the 6 bp consensus EarA-binding sequence upstream of the fla promoter in the P. furiosus genome.

Pontibacterium granulatum gen. nov., sp. nov., isolated from a tidal flat.

A Gram-stain-negative, strictly aerobic, moderately halophilic bacterium, designated A-1T, was isolated from a tidal flat of the Taean coast in South Korea. Cells were motile rods with a single flagellum showing oxidase-negative and catalase-positive activities and contained poly-ß-hydroxyalkanoic acid granules. Growth of strain A-1T was observed at 20-40 °C (optimum, 30 °C), pH 6.0-10.5 (optimum, pH 7.0) and in the presence of 1.0-6.0 % (w/v) NaCl (optimum, 2.0 %). Strain A-1T contained C16 : 0, summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c) and summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c) as the major fatty acids. The major polar lipids of strain A-1T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The isoprenoid quinones detected were ubiquinone-7 and ubiquinone-8. The G+C content of the genomic DNA was 51.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain A-1T formed a distinct phylogenetic lineage from other genera within the family Oceanospirillaceae. Strain A-1T shared low 16S rRNA gene sequence similarities with other taxa (≤94.9 %). On the basis of phenotypic, chemotaxonomic and molecular properties, it is clear that strain A-1T represents a novel genus and species of the family Oceanospirillaceae, for which the name Pontibacterium granulatum gen. nov., sp. nov. is proposed. The type strain is A-1T (=KACC 18119T=JCM 30136T).

Identification of a gene encoding a membrane-anchored toll-like receptor 5 (TLR5M) in Oplegnathus fasciatus that responds to flagellin challenge and activates NF-κB.

Toll-like receptor 5 (TLR5) recognizes bacterial flagellin and induces the downstream signaling through the myeloid differentiation primary response gene 88 (MyD88) protein to produce proinflammatory cytokines. In this study, we describe a TLR5 membrane form (OfTLR5M) and its adaptor protein MyD88 (OfMyD88) in rock bream, Oplegnathus fasciatus. Both Oftlr5m (6.7 kb) and Ofmyd88 (3.7 kb) genes displayed a quinquepartite structure with five exons and four introns. Protein structure of OfTLR5M revealed the conventional architecture of TLRs featured by an extracellular domain with 22 leucine rich repeats (LRR), a transmembrane domain and an endodomain with TIR motif. Primary OfTLR5M sequence shared a higher homology with teleost TLR5M. The evolutional analysis confirmed that TLR5 identified in the current study is a membrane receptor and the data further suggested the co-evolution of the membrane-anchored and soluble forms of TLR5 in teleosts. Inter-lineage comparison of gene structures in vertebrates indicated that the tlr5m gene has evolved with extensive rearrangement; whereas, the myd88 gene has maintained a stable structure throughout the evolution. Inspection of 5' flanking region of these genes disclosed the presence of several transcription factor binding sites including NF-κB. Quantitative real-time PCR (qPCR) detected Oftlr5m mRNA in eleven tissues with the highest abundance in liver. In vivo flagellin administration strongly induced the transcripts of both Oftlr5m and Ofmyd88 in gills and head kidney tissues suggesting their ligand-mediated upregulation. In a luciferase assay, HEK293T cells transiently transfected with Oftlr5m and Ofmyd88 demonstrated a higher NF-κB activity than the mock control, and the luciferase activity was intensified when cells were stimulated with flagellin. Collectively, our study represents the genomic, evolutional, expressional and functional insights into a receptor and adaptor molecules of teleost origin that are involved in flagellin sensing.

Flagellar adhesion in Trypanosoma brucei relies on interactions between different skeletal structures in the flagellum and cell body.

The Trypanosoma brucei flagellum is an essential organelle anchored along the surface of the cell body through a specialized structure called the flagellum attachment zone (FAZ). Adhesion relies on the interaction of the extracellular portion of two transmembrane proteins, FLA1 and FLA1BP. Here, we identify FLAM3 as a novel large protein associated with the flagellum skeleton whose ablation inhibits flagellum attachment. FLAM3 does not contain transmembrane domains and its flagellar localization matches closely, but not exactly, that of the paraflagellar rod, an extra-axonemal structure present in the flagellum. Knockdown of FLA1 or FLAM3 triggers similar defects in motility and morphogenesis, characterized by the assembly of a drastically reduced FAZ filament. FLAM3 remains associated with the flagellum skeleton even in the absence of adhesion or a normal paraflagellar rod. However, the protein is dispersed in the cytoplasm when flagellum formation is inhibited. By contrast, FLA1 remains tightly associated with the FAZ filament even in the absence of a flagellum. In these conditions, the extracellular domain of FLA1 points to the cell surface. FLAM3 is essential for proper distribution of FLA1BP, which is restricted to the most proximal portion of the flagellum upon knockdown of FLAM3. We propose that FLAM3 is a key component of the FAZ connectors that link the axoneme to the adhesion zone, hence it acts in an equivalent manner to the FAZ filament complex, but on the side of the flagellum.

Resveratrol enhances TNF-α production in human monocytes upon bacterial stimulation.

BACKGROUND: Resveratrol is a key component of red wine that has been reported to have anti-carcinogenic and anti-aging properties. Additional studies conducted in vitro and in animal models suggested anti-inflammatory properties. However, data from primary human immune cells and in vivo studies are limited. METHODS: A pilot study was performed including 10 healthy volunteers. Plasma cytokine levels were measured over 48h after oral application of 5g resveratrol. To verify the in vivo findings, cytokine release and gene expression in human peripheral blood mononuclear cells (PBMC) and/or monocytes was assessed after treatment with resveratrol or its metabolites and stimulation with several toll-like receptor (TLR)-agonists. Additionally, the impact on intracellular signaling pathways was analyzed using a reporter cell line and Western blotting. RESULTS: Resveratrol treated individuals showed a significant increase in tumor necrosis factor-α (TNF-α) levels 24h after treatment compared to baseline. Studies using human PBMC or isolated monocytes confirmed potentiation of TNF-α production with different TLR agonists, while interleukin (IL)-10 was inhibited. Moreover, we observed significantly enhanced nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) activation using a reporter cell line and found increased phosphorylation of p105, which is indicative of alternative NF-κB pathway activation. GENERAL SIGNIFICANCE: By administering resveratrol to healthy humans and utilizing primary immune cells we were able to detect TNF-α enhancing properties of the agent. In parallel, we found enhanced alternative NF-κB activation. We report on a novel pro-inflammatory property of resveratrol which has to be considered in concepts of its biologic activity.

The fasciclin-like arabinogalactan protein family of Eucalyptus grandis contains members that impact wood biology and biomechanics.

Fasciclin-like arabinogalactan protein (FLA) families have been identified and characterised in key plant species, with some members exhibiting functional specialization. Here we identify the FLA family of Eucalyptus grandis, and investigate the roles of three single-FAS domain FLAs, with particular focus on secondary cell-wall formation and wood properties. We use various in-silico approaches to identify and characterise E. grandis genome FLAs, and perform phylogenetic comparisons with other species. For three key FLAs, we perform functional testing including promoter-reporter and overexpression transgenic approaches using eucalypts, poplar and tobacco. Of the 18 eucalypt FLAs identified, several were specifically and highly expressed in stems. The specificity to stem xylem vessel and fibre development was demonstrated with EniFLA1promoter:GUS studies in several species. Testing of select eucalypt FLAs resulted in altered wood development and properties, for example 35S:EgrFLA2 led to a 3 degree reduction in cellulose microfibril angle in eucalypt xylem fibres, and 35S:EgrFLA3 to a reduction in tobacco stem flexural strength. These results indicate that the eucalypt FLA family contains diverse members, and particular members with single FAS domains that are functionally specialized for secondary cell wall growth and properties.

Insights into molecular profiles and genomic evolution of an IRAK4 homolog from rock bream (Oplegnathus fasciatus): immunogen- and pathogen-induced transcriptional expression.

As a pivotal signaling mediator of toll-like receptor (TLR) and interleukin (IL)-1 receptor (IL-1R) signaling cascades, the IL-1R-associated kinase 4 (IRAK4) is engaged in the activation of host immunity. This study investigates the molecular and expressional profiles of an IRAK4-like homolog from Oplegnathus fasciatus (OfIRAK4). The OfIRAK4 gene (8.2 kb) was structured with eleven exons and ten introns. A putative coding sequence (1395bp) was translated to the OfIRAK protein of 464 amino acids. The deduced OfIRAK4 protein featured a bipartite domain structure composed of a death domain (DD) and a kinase domain (PKc). Teleost IRAK4 appears to be distinct and divergent from that of tetrapods in terms of its exon-intron structure and evolutionary relatedness. Analysis of the sequence upstream of translation initiation site revealed the presence of putative regulatory elements, including NF-κB-binding sites, which are possibly involved in transcriptional control of OfIRAK4. Quantitative real-time PCR (qPCR) was employed to assess the transcriptional expression of OfIRAK4 in different juvenile tissues and post-injection of different immunogens and pathogens. Ubiquitous basal mRNA expression was widely detected with highest level in liver. In vivo flagellin (FLA) challenge significantly intensified its mRNA levels in intestine, liver and head kidney indicating its role in FLA-induced signaling. Meanwhile, up-regulated expression was also determined in liver and head kidney of animals challenged with potent immunogens (LPS and poly I:C) and pathogens (Edwardsiella tarda and Streptococcus iniae and rock bream iridovirus (RBIV)). Taken together, these data implicate that OfIRAK4 might be engaged in antibacterial and antiviral immunity in rock bream.

Molecular genomic- and transcriptional-aspects of a teleost TRAF6 homolog: Possible involvement in immune responses of Oplegnathus fasciatus against pathogens.

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a crucial docking molecule for TNFR superfamily and Interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily. As an adaptor protein in pathogen-induced signaling cascades, TRAF6 modulates both adaptive- and innate-immunity. In order to understand the immune responses of teleost TRAF6, Oplegnathus fasciatus TRAF6-like gene (OfTRAF6) was identified and characterized. Genomic length of OfTRAF6 (4 kb), obtained by means of a genomic BAC library, spanned seven exons which represented a putative coding sequence of 1716 bp and encoded 571 amino acids (aa) with an estimated molecular weight of 64 kDa. This putative protein demonstrated the classical tetra-domain architecture composed of a zinc finger RING-type profile, two zinc finger TRAF-type profiles, a coiled-coil region and a MATH domain. While the sequence similarity with human TRAF6 was 66.5%, OfTRAF6 shared a higher overall similarity with teleost homologs (∼75-92%). Phylogeny of TRAF-family was examined and TRAF6-subfamily appeared to be the precursor of other subfamilies. In addition, the clustering pattern confirmed that OfTRAF6 is a novel member of TRAF6subfamily. Based on comparative genomic analysis, we found that vertebrate TRAF6 exhibits two distinct structures in teleost and tetrapod lineages. An intron-loss event has probably occurred in TRAF6 gene during the evolution of tetrapods from teleosts. Inspection of putative OfTRAF6 promoter revealed the presence of several immune responsive transcription factor binding sites. Real-time qPCR assay detected OfTRAF6 transcripts in eleven juvenile fish tissues with higher levels in peripheral blood cells followed by liver. Putative role of OfTRAF6 in response to flagellin, LPS, poly I:C, pathogenic bacteria (Edwardsiella tarda and Streptococcus iniae) and rock bream iridovirus (RBIV) was profiled in different tissues and OfTRAF6 revealed up-regulated transcript levels. Altogether, these findings implicate that OfTRAF6 is not only involved in flagellin-induced signaling cascade, but also contributes to the antibacterial- and antiviral-responses.

Gene structure, molecular characterization and transcriptional expression of two p38 isoforms (MAPK11 and MAPK14) from rock bream (Oplegnathus fasciatus).

The p38 kinases are one of the four subgroups of mitogen-activated protein kinase (MAPK) superfamily which are involved in the innate immunity. The p38 subfamily that includes four members namely p38α (MAPK14), p38ß (MAPK11), p38γ (MAPK12) and p38δ (MAPK13), regulates the activation of several transcription factors. In this study, a p38ß (OfMAPK11) homolog and a p38α (OfMAPK14) homolog of Oplegnathus fasciatus were identified at genomic level. Results clearly showed that both MAPK11 and MAPK14 are well-conserved at both genomic structural- and amino acid (aa)-levels. Genomic sequences of OfMAPK11 (∼ 15.6 kb) and OfMAPK14 (∼ 13.4 kb) had 12 exons. A comparison of exon-intron structural arrangement of these genes from different vertebrate lineages indicated that all the exon lengths are highly conserved, except their terminal exons. Full-length cDNAs of OfMAPK11 (3957 bp) and OfMAPK14 (2504 bp) encoded corresponding proteins of 361 aa and 360 aa, respectively. Both OfMAPK proteins harbored a Ser/Thr protein kinases catalytic domain (S_TKc domain) which includes an activation loop with a dual phosphorylation site (TGY motif) and several specific-binding sites for ATP and substrates. Molecular modeling of the activation loop and substrate binding sites of rock bream MAPKs revealed the conservation of crucial residues and their orientation in 3D space. Transcripts of OfMAPKs were ubiquitously detected in eleven tissues examined, however at different levels. The modulation of OfMAPKs' transcription upon pathogen-associated molecular patterns (PAMPs: flagellin, lipopolysaccharide and poly I:C) and pathogens (Edwardsiella tarda, Streptococcus iniae and rock bream iridovirus) was investigated. Among the seven examined tissues, the flagellin-challenge upregulated the mRNA level of both OfMAPKs in the head kidney. Meanwhile, modulation of OfMAPK mRNA expression in the liver upon other immune-challenges varied in a time-dependent manner. Collectively, these results suggest that OfMAPKs are true members of p38 subfamily, which might be induced by different immune stimuli.