IRF11 synergizes with STAT1 and STAT2 to promote type I IFN production.

Interferon regulatory factor 11 (IRF11), a fish specific member of IRF family, is a transcription factor known for its positive role in teleost antiviral defense by regulating IFN expression. Despite its recognized function, the precise mechanism of IRF11 in type I IFNs production remains largely unknown. In this study, we identified IRF11 in Japanese eel, Anguilla japonica, (AjIRF11) and determined its involvement in the later phase of fish IFN production. Our results demonstrate that IRF11-induced IFN production operates through ISRE binding. Mutations in each ISRE site within the promoter of AjIFN2 or AjIFN4 abolished IRF11-mediated activation of IFN promoters. In addition, the overexpression of AjIRF11 does not significantly impact the activation of AjIFN promoters induced by RLR-related signaling pathway proteins. Furthermore, IRF11-knockdown in ZFLs (zebrafish liver cells) has no effect on the RLRs-induced expression of zebrafish IFN-φ1 and IFN-φ3, indicating that IRF11 is not involved in the RLR-mediated IFN production. However, AjIRF11 can form transcription complexes with AjSTAT1 or AjSTAT2, or form homo- or heterodimers with AjIRF1 to stimulate the transcription of type I IFNs. Overall, it is shown in this study that IRF11 can act synergistically with STAT1 and/or STAT2 for the induction of IFN.

Iruplinalkib (WX­0593), a novel ALK/ROS1 inhibitor, overcomes crizotinib resistance in preclinical models for non-small cell lung cancer.

Despite remarkable initial responses of anaplastic lymphoma kinase (ALK) inhibitors in ALK-positive non-small cell lung cancer (NSCLC) patients, cancers eventually develop resistance within one to two years. This study aimed to compare the properties of iruplinalkib (WX­0593) with other ALK inhibitors and report the comprehensive characterization of iruplinalkib against the crizotinib resistance. The inhibitory effect of iruplinalkib on kinase activity was detected. A kinase screen was performed to evaluate the selectivity of iruplinalkib. The effect of iruplinalkib on related signal transduction pathways of ALK and c-ros oncogene 1 (ROS1) kinases was examined. The cellular and in vivo activities of ALK inhibitors were compared in engineered cancer-derived cell lines and in mice xenograft models, respectively. Human hepatocytes derived from three donors were used for evaluating hepatic enzyme inducing activity. HEK293 cell lines expressing transportors were used to invesigated the drug interaction potential mediated by several transporters. The results showed iruplinalkib potently inhibited the tyrosine autophosphorylation of wild-type ALK, ALKL1196M, ALKC1156Y and epidermal growth factor receptor (EGFR)L858R/T790M. The inhibitory effects of iruplinalkib in patient-derived xenograft and cell line-derived xenograft models were observed. Moreover, iruplinalkib showed robust antitumor effects in BALB/c nude mice xenograft models with ALK-/ROS1-positive tumors implanted subcutaneously, and the tumor suppressive effects in crizotinib-resistant model was significantly better than that of brigatinib. Iruplinalkib did not induce CYP1A2, CYP2B6 and CYP3A4 at therapeutic concentration, and was also a strong inhibitor of MATE1 and MATE2K transporters, as well as P-gp and BCRP. In conclusion, iruplinalkib, a highly active and selective ALK/ROS1 inhibitor, exhibited strong antitumor effects in vitro and in crizotinib-resistant models.

Molecular Characterization of the Von Willebrand Factor Type D Domain of Vitellogenin from Takifugu flavidus.

The von Willebrand factor type D (VWD) domain in vitellogenin has recently been found to bind tetrodotoxin. The way in which this protein domain associates with tetrodotoxin and participates in transporting tetrodotoxin in vivo remains unclear. A cDNA fragment of the vitellogenin gene containing the VWD domain from pufferfish (Takifugu flavidus) (TfVWD) was cloned. Using in silico structural and docking analyses of the predicted protein, we determined that key amino acids (namely, Val115, ASP116, Val117, and Lys122) in TfVWD mediate its binding to tetrodotoxin, which was supported by in vitro surface plasmon resonance analysis. Moreover, incubating recombinant rTfVWD together with tetrodotoxin attenuated its toxicity in vivo, further supporting protein-toxin binding and indicating associated toxicity-neutralizing effects. Finally, the expression profiling of TfVWD across different tissues and developmental stages indicated that its distribution patterns mirrored those of tetrodotoxin, suggesting that TfVWD may be involved in tetrodotoxin transport in pufferfish. For the first time, this study reveals the amino acids that mediate the binding of TfVWD to tetrodotoxin and provides a basis for further exploration of the molecular mechanisms underlying the enrichment and transfer of tetrodotoxin in pufferfish.

Macrophage migration inhibitory factor (MIF) family in arthropods: Cloning and expression analysis of two MIF and one D-dopachrome tautomerase (DDT) homologues in mud crabs, Scylla paramamosain.

The macrophage migration inhibitory factor (MIF) family, consisting of MIF and D-dopachrome tautomerase (DDT) in vertebrates, is evolutionarily ancient and has been found across Kingdoms including vertebrates, invertebrates, plants and bacteria. The mammalian MIF family are chemokines at the top of the inflammatory cascade in combating infections. They also possess enzymatic activities, e.g. DDT catalysis results in the production of 5,6-dihydroxyindole (DHI), a precursor of eumelanin. MIF-like genes are widely distributed, but DDT-like genes have only been described in vertebrates and a nematode. In this report, we cloned a DDT-like gene, for the first time in arthropods, and a second MIF in mud crab. The mud crab MIF family have a three exon/two intron structure as seen in vertebrates. The identification of a DDT-like gene in mud crab and other arthropods suggests that the separation of MIF and DDT preceded the divergence of protostomes and deuterostomes. The MIF family is differentially expressed in tissues of adults and during embryonic development and early life. The high level expression of the MIF family in immune tissues, such as intestine and hepatopancreas, suggests an important role in mud crab innate immunity. Mud crab DDT is highly expressed in early embryos, in megalops and crablets and this coincides with the requirement for melanisation in egg chorion tanning and cuticular hardening in arthropods, suggesting a potential novel role of DDT in melanogenesis via its tautomerase activity to produce DHI in mud crab. The clarification of the presence of both MIF and DDT in this report paves the way for further investigation of their functional roles in immunity and in melanogenesis in mud crab and other arthropods.

A new antimicrobial peptide SCY2 identified in Scylla Paramamosain exerting a potential role of reproductive immunity.

A new antimicrobial peptide named SCY2 with 65.08% identity in amino acid sequence to the known scygonadin (SCY1) was first characterized in Scylla paramamosain based on its cloned full-length cDNA and genomic DNA sequences. The SCY2 gene was dominantly expressed in the ejaculatory duct of male crabs and its mRNA transcripts were discerned mainly in the glandular epithelium of the inner wall and the secretion inside the ejaculatory duct. Although the SCY2 gene could not be induced with the challenge of the bacteria and fungi tested, its induction reached the highest level at the peak period of mating in mature male crabs either in June or November, suggesting its induction was likely related to seasonal reproduction changes. Moreover, it was interesting to note that, from analysis of its transcripts and protein, SCY2 was significantly expressed only in the ejaculatory duct of pre-copulatory males before mating, however it was clearly detected in the spermatheca of post-copulatory females after mating accompanied by the decreased level of SCY2 expression in the ejaculatory duct. These results suggested that the SCY2 was probably transferred from the male during mating action with the female for the purpose of protecting fertilization. The recombinant SCY2 was more active against the Gram-positive than the Gram-negative bacteria tested. It was further observed that the SCY2 transcripts were significantly increased with addition of exogenous progesterone in tissue cultures whereas the several other hormones tested had no any effect on SCY2 expression, indicating that there might be a relationship between the SCY2 expression and the induction of hormones in vivo. In summary, this study demonstrated that one role of SCY2 was likely to be involved in crab reproduction and it exerted its reproductive immune function through the mating action and the maintenance of inner sterility in the spermatheca of the female, thus leading to successful fertilization of S. paramamosain.

Flagellar motility contributes to the invasion and survival of Aeromonas hydrophila in Anguilla japonica macrophages.

The interaction between pathogenic bacteria and the host phagocytes is complicated. It is generally believed that only obligate intracellular pathogens can invade and survive in host phagocytes. In this study, we revealed that the pathogenic Aeromonas hydrophila B11 can also invade and survive in the macrophages of its host Anguilla japonica in vitro. To further investigate the mechanisms of A. hydrophila invasion and survival in host macrophages, a mini-Tn10 transposon mutagenesis system was used to generate an insertion mutant library by cell conjugation between the donor Escherichia coli Sm10 (pLOFKm) and the recipient A. hydrophila B11. Out of 465 individual colonies, 13 mutants impaired in survival within macrophages were selected, and the mutant BM116 was the most seriously impaired strain. Molecular analysis showed that an ORF of approximately 1335 bp (GenBank accession numbers JQ974982) of the mutant BM116 was inserted by mini-Tn10. This ORF putatively encodes a deduced 445 amino acids protein that displays the highest identity (99.6%) with the flagellar hook protein FlgE of A. hydrophila subsp. hydrophila ATCC 7966. The biological characteristics of the wild-type B11, the mutant B116 and the complemented strain were investigated. The results reveal that the flagella of the mutant BM116 was absent and that these mutant bacteria exhibited defective motility, adhesion, and invasion and survival in host macrophages when compared with the wild type and the complemented strain. These findings indicate that flgE is required for flagellum biogenesis in A. hydrophila and that flagellar motility is required for A. hydrophila invasion and survival in the macrophages of its host. Our findings provide an important new understanding of the nonintracellular pathogenic bacteria invasion and survival in host phagocytes and the interactions between the pathogens and their host.

Myeloid-derived Wnts play an indispensible role in macrophage and fibroblast activation and kidney fibrosis.

Wnt/ß-catenin signaling plays a pivotal role in the pathogenesis of chronic kidney diseases (CKD), which is associated with macrophage activation and polarization. However, the relative contribution of macrophage-derived Wnts in the evolution of CKD is poorly understood. Here we demonstrate a critical role of Wnts secreted by macrophages in regulating renal inflammation and fibrosis after various injuries. In mouse model of kidney fibrosis induced by unilateral ureteral obstruction (UUO), macrophages were activated and polarized to M1 and M2 subtypes, which coincided with the activation of Wnt/ß-catenin signaling. In vitro, multiple Wnts were induced in primary cultured bone marrow-derived macrophages (BMDMs) after polarization. Conversely, Wnt proteins also stimulated the activation and polarization of BMDMs to M1 and M2 subtype. Blockade of Wnt secretion from macrophages in mice with myeloid-specific ablation of Wntless (Wls), a cargo receptor that is obligatory for Wnt trafficking and secretion, blunted macrophage infiltration and activation and inhibited the expression of inflammatory cytokines. Inhibition of Wnt secretion by macrophages also abolished ß-catenin activation in tubular epithelium, repressed myofibroblast activation and reduced kidney fibrosis after either obstructive or ischemic injury. Furthermore, conditioned medium from Wls-deficient BMDMs exhibited less potency to stimulate fibroblast proliferation and activation, compared to the controls. These results underscore an indispensable role of macrophage-derived Wnts in promoting renal inflammation, fibroblasts activation and kidney fibrosis.

The origin and loss of interferon regulatory factor 10 (IRF10) in different lineages of vertebrates.

The vertebrate IFN regulatory factor (IRF) family consists of 11 members that exert distinct roles in a variety of biological processes, including antiviral defense, regulation of cell proliferation, differentiation and apoptosis. Of these, IRF10 is widely present in different vertebrate lineages, but appears to have been lost in primates and rodents. To understand the evolutionary occurrence of IRF10, we performed comparative analyses of currently available genomic data in a taxonomically diverse set of vertebrates, and found that IRF10 originated after the divergence of chondrichthyans from gnathostomes. Phylogenetically, vertebrate IRF10 is much more closely related to IRF4 than to IRF8 or IRF9, although these four IRFs may have a common ancestor. In addition, the loss of IRF10 in Euarchontoglires might be resulted from mutation accumulation, and the rate of mutations in rodents appears to be higher than in the primate lineage. In primates, the gene-disruptive mutations may have occurred at least prior to the separation of new world monkey and old world primates, roughly 40 million years ago. Overall, we propose a detailed evolutionary scenario for IRF10, which may help us understand the evolutionary mechanisms in the expansion and contraction of the IRF family.

Nuclear import of IRF11 via the importin α/ß pathway is essential for its antiviral activity.

Interferon regulatory factor 11 (IRF11), an intriguing IRF member found only in fish species, has recently been shown to have antiviral properties that are dependent on its nuclear entry and DNA binding affinity. However, the mechanisms by which IRF11 enters the nucleus are unknown. In the present study, we found orthologs of IRF11 in lamprey and lancelet species by combining positional, phylogenetic and structural comparison data, showing that this gene has an ancient origin. The IRF11 gene (AjIRF11) from the Japanese eel, Anguilla japonica, was subsequently characterized, and it was found that AjIRF11 has antiviral activities against spring viremia of carp virus (SVCV), which are accomplished by regulating the production of type I IFN and IFN-stimulated genes. In addition to its known DNA binding residues in the α3 helix, two residues in Loop 1, His40 and Trp46, are also involved in DNA binding and activation of the IFN promoter. Using immunofluorescence microscopy and site-directed mutagenesis analysis, we confirmed that full nuclear localization of AjIRF11 requires the bipartite nuclear localization sequence (NLS) spanning residues 75 to 101, as well as the monopartite NLS situated between residues 119 and 122. Coimmunoprecipitation assays confirmed that AjIRF11 interacts with importin α via its NLSs and can also bind to importin ß directly, implying that IRF11 can be imported to the nucleus by one or more transport receptors.

Effects of weaning American glass eels (Anguilla rostrata) with the formula diet on intestinal microbiota and inflammatory cytokines genes expression.

This study aimed to investigate the effects of weaning American glass eels (Anguilla rostrata) with the formula diet on intestinal microbiota and the expression of inflammatory cytokines genes. During the feeding trial, the control group (termed IF group) was fed with initial feed for 34 days, and the experimental group (termed FF group) was fed with initial feed for 30 days, and then weaned with the formula diet for 4 days. After feeding trial, intestines were subjected to microbiota analysis using 16S rDNA high-throughput sequencing, and expression of three inflammatory cytokines genes in gut were examined by qPCR. The results indicated that the species richness and diversity of intestinal microbiota exhibited significantly higher in FF group than that in IF group (P < 0.05). At the phylum level, the core intestinal microflora was the same for two groups. The most abundant phylum was Firmicutes in IF group, while it was Proteobacteria in FF group. Five genera were significantly higher in the IF group compared with the FF group, and Bacillus was the most major enriched biomarker at genus level. Nine genera were significantly higher in the FF group compared with the IF group, and Acidovorax was the most major enriched biomarker. Weaning from initial feeding diet to formula feeding diet enhanced the expression levels of TNF-α and IL-8, and there was no significant change in IL-1ß expression between the two groups. These findings would be very useful to improve the diet formulation for weaning stage of American glass eels.

Two copies of the genes encoding the subunits of putative interleukin (IL)-4/IL-13 receptors, IL-4Rα, IL-13Rα1 and IL-13Rα2, have been identified in rainbow trout (Oncorhynchus mykiss) and have complex patterns of expression and modulation.

Mammalian interleukin-4 (IL-4) and IL-13 are T helper type 2 (Th2) cytokines with pleiotropic functions in immunity. They signal through receptors containing IL-4Rα and IL-2Rγ or IL-13Rα1. In addition, a decoy receptor, IL-13Rα2, is known to exist and modulates the function of IL-13. The existence of fish orthologues to mammalian IL-4 and IL-13 is still under debate. However, the receptor chains have been predicted in zebrafish, and we have previously cloned IL-2Rγ and IL-13Rα2 in rainbow trout. In this study, we have cloned a further five novel trout IL-4/13 receptors. Thus, each of the IL-4Rα, IL-13Rα1 and IL-13Rα2 chains has two copies. The identities of the receptors is supported by homology analysis, characteristic domain structure, phylogenetic tree analysis and synteny analysis in zebrafish. However, the characteristic WSXWS motif of structural importance in mammalian type I cytokine receptors is missing in all fish IL-4Rα and IL-13Rα1 molecules. All the receptors have a characteristic domain structure that is similar to their mammalian counterparts except for IL-13Rα1b that has the N-terminal Ig domain missing. Since this Ig domain is a specific and critical binding unit for IL-13 but not for IL-4 signalling, its absence potentially converts the IL-13Rα1b into a receptor that can only signal via IL-4 ligation. The existence of duplicated receptor genes perhaps suggests that more ligands still remain to be discovered that will bind these receptors. The duplicated receptors are differentially expressed in most tissues and cell lines examined, and their expression can be modulated by LPS, polyIC and IFN-γ in cell lines. In contrast, the T-cell stimulant phytohaemagglutinin increased the expression of IL-4Rα1 and IL-4Rα2, but not IL-13Rα1/2, suggesting a role of an IL-4-like molecule in T-cell growth/activation in fish.

The gamma-chain cytokine/receptor system in fish: more ligands and receptors.

The mammalian gamma-chain (γC) cytokine family consists of interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21. They signal through a receptor complex containing the common γC and a private alpha chain, and in the case of IL-2 and IL-15 an additional common IL-2/15Rß chain. Deficiency of γC signalling in mammals prevents CD4+ T cells from developing effector functions and CD8+ T cells from developing immunological memory. Thus γC cytokines are critical for the generation and peripheral homeostasis of naïve and memory T cells. This review will give an update on the γC ligands and receptor subunits in fish, and also present some new data on the cloning and expression of a second γC and two IL-2Rß chains in rainbow trout Oncorhynchus mykiss. In recent years, aided by the availability of sequenced fish genomes and expressed sequence tag databases, five of the six mammalian γC cytokines and their cognate receptors have been discovered in fish, with only the IL-9/IL-9R homologues apparently absent. Paralogues have been discovered in diploid fish and all the receptors described in the tetraploid rainbow trout, including γC itself, IL-2Rß, IL-4Rα, IL-13Rα1, IL-13Rα2 and IL-2/15Rα, have duplicates. As a consequence of the teleost and salmonid whole genome duplications, even more paralogues may yet be discovered. Some of the paralogues have changes in domain structures and show differential expression and modulation, suggesting the potential for a change in function. Functional characterisation of fish γC cytokines is beginning but made more difficult by the co-existence of so many paralogues of the ligands and their receptors. Initial functional studies have shown that fish γC cytokines can modulate the expression of key cytokines (e.g. interferon-γ, IL-10 and IL-22) of the adaptive immune response, and may thus have promise as adjuvants to improve vaccination efficiency in fish.

Quantitative gene expression and in situ localization of scygonadin potentially associated with reproductive immunity in tissues of male and female mud crabs, Scylla paramamosain.

Scygonadin (Scy) is an important antimicrobial peptide which was first isolated from the seminal plasma of Scylla serrata (now renamed as Scylla paramamosain). Elucidation of the Scy expression pattern in tissues will help in understanding its potential function associated with the reproductive immunity. In our study, Scy mRNA transcripts and its protein were found widely distributed in mature male and female crabs. Scy mRNA transcripts were significantly demonstrated in the ejaculatory duct and hemocytes of males but were much less expressed in the other tissues tested. In addition, Scy mRNA transcripts were discerned in a number of cells in the glandular epithelium of the inner wall and in the secretion inside the ejaculatory duct using the in situ hybridization method. In females, Scy mRNA transcripts were obviously demonstrated in the hemocytes and gills but weakly detected in other tissues tested. The copy number of scygonadin mRNA transcripts in the ejaculatory duct of males was greatly higher than those in other tissues, in particular, was over 60,000 fold that in the hemocytes of females. Using immunohistochemistry, the Scy protein was found at higher levels in male tissues than in female ones, particularly in the reproductive duct of males. It was also interesting to note that Scy gene expression was not significantly induced with lipopolysaccharide challenge. However, it was highly expressed in the ejaculatory duct and the seminal vesicle of pre-copulatory males and in the spermathecae of post-copulatory females under mating conditions. The results suggested that Scy, as an important antimicrobial component, probably performed more functions in males, and was likely to be involved in a function associated with crab fertilization and reproduction in both males and females during mating.

Enhancement of protective immunity in European eel (Anguilla anguilla) against Aeromonas hydrophila and Aeromonas sobria by a recombinant Aeromonas outer membrane protein.

To develop a vaccine, which can simultaneously prevent the diseases caused by various pathogenic bacteria in fish, we try to find a conserved outer membrane protein (OMP) antigen from different bacterial pathogens. In this study, an OMP fragment of 747 bp (named as Omp-G), which was highly conserved in seven Aeromonas OMP sequences from the NCBI database, was amplified by PCR from one Aeromonas sobria strain (B10) and two Aeromonas hydrophila strains (B27 and B33) with the designed specific primers. The sequence was cloned into pGEX-2T (6 × His-tag) vector, expressed in Escherichia coli system, and then the recombinant protein (named as rOmp-G) was purified with nickel chelating affinity chromatography. The purified rOmp-G showed a good immunogenicity in rabbits and well-conserved characteristics in these three pathogens by enzyme-linked immunosorbed assay. Furthermore, the rOmp-G also showed good immunogenicity in eels (Anguilla anguilla) for eliciting significantly increased specific antibodies (P < 0.01), and providing higher protection efficiencies (P < 0.05) after the pathogens challenge. The values of the relative percent survival in eels were 70% and 50% for two A. hydrophila strain challenge, and 75% for A. sobria strain challenge. This is the first report of a potential vaccination in eels that simultaneously provide protectiveness against different Aeromonas pathogens with a conserved partial OMP.

Functional domains and amino acid residues of Japanese eel IRF1, AjIRF1, regulate its nuclear import and IFN/Mx promoter activation.

Interferon regulatory factors (IRFs) are a family of transcriptional factors capable of regulating the expression of distinct subsets of interferon (IFN)-stimulated genes by binding to their promoters. IRF1 was the first member identified for its ability to regulate the IFNß gene and has now been revealed to exhibit remarkable functional diversity in the regulation of different cellular responses. In the present study, the IRF1 gene was identified and characterized in Japanese eel, Anguilla japonica (AjIRF1). The open reading frame of AjIRF1 was 804 bp in length, encoding a protein of 267 amino acids (aa) that encompasses a conserved N-terminal DNA binding domain (DBD). Sequence alignment shows the presence of six highly conserved tryptophan (W) residues in the DBD of IRF1, IRF2 and IRF11, while other IRF members have only five tryptophans. Expression analysis showed that AjIRF1 was significantly upregulated in all tested organs/tissues in response to Poly I:C stimulation or Edwardsiella tarda infection. Furthermore, the functional activity of AjIRF1 was confirmed in driving the transcription of AjIFN promoters, which depends on the highly conserved residues within DBD. Subcellular distribution analysis revealed that AjIRF1 was localized exclusively in the nucleus, which is cooperatively regulated by a bipartite NLS embedded within the DBD and a monopartite NLS located immediately downstream of the DBD. Taken together, this study presents the expression profile of AjIRF1 and defines the functional motifs required for its nuclear import and its role in activating IFN promoters, thus providing helpful information for further research on the regulatory mechanisms of teleost IRF1.

Molecular insight, expression profile and subcellular localization of two STAT family members, STAT1a and STAT2, from Japanese eel, Anguilla japonica.

Signal transducer and activator of transcription 1 (STAT1) and STAT2 are critical components of type I and type II IFNs signaling. To date, seven STAT family proteins have been identified from mammals. However, the information on STAT genes in teleost fish is still limited. In the present study, two STAT family genes (STAT1a and STAT2) were identified from Japanese eel, Anguilla japonica and designated as AjSTAT1a and AjSTAT2. The open reading frames of AjSTAT1a and AjSTAT2 are 2244 bp and 2421 bp, encoding for polypeptides of 747 aa and 806 aa, respectively. Both AjSTAT1a and AjSTAT2 contain the conserved domains of STAT proteins. Phylogenetic analysis was performed based on the STATs protein sequences, and showed that AjSTAT1a and AjSTAT2 shared the closest relationship with Oncorhynchus mykiss. Quantitative real-time PCR analysis revealed that AjSTAT1a and AjSTAT2 were expressed in most examined tissues, with the highest expression both in blood. Significantly up-regulated transcripts of AjSTAT1a and AjSTAT2 were detected in response to poly I:C stimulation, and Edwardsiella tarda induced increase in the expression of AjSTAT1a and AjSTAT2 genes. Subcellular localization analysis showed that in both IFNγ-stimulated and unstimulated EPC cells AjSTAT1a and AjSTAT2 were mainly distributed in the cytoplasm, but few AjSTAT1a was distributed in the nucleus. All these results suggested that AjSTAT1a and AjSTAT2 may be critical for regulating the host innate immune defense against pathogens invasion.

Soluble expression and purification of a crab antimicrobial peptide scygonadin in different expression plasmids and analysis of its antimicrobial activity.

Scygonadin is an anionic antimicrobial peptide recently identified from the seminal plasma of Scylla serrata. To gain more detailed information on its antimicrobial activity, scygonadin mature peptide was expressed in Escherichia coli in order to obtain a large quantity of biologically active product. An approximately 43 kDa fusion protein CKS-scygonadin was obtained in a highly stable and soluble form. The soluble component of the fusion CKS-scygonadin was purified by immobilized metal affinity chromatography (IMAC). A single 11 kDa recombinant scygonadin was cleaved from CKS-scygonadin and purified from the cleavage mixture using an affinity chromatography column with a yield of 10.6 mg/L. Alternatively, a recombinant scygonadin was purified from pET28-scygonadin by one-step Ni(2+) affinity chromatography and 65.9 mg/L pure recombinant scygonadin was obtained which was higher than that purified from pTrc-CKS/scygonadin in bacteria culture. The recombinant scygonadin was confirmed using SDS-PAGE analysis and MS-fingerprinting. Both recombinant products of scygonadin from different expressed plasmids showed the activity against both Gram-positive and Gram-negative bacteria, but no activity against yeast and fungi tested. The kinetic studies showed that the recombinant scygonadin was strong active against Staphylococcus aureus and the killing of S. aureus appeared time and dose dependent. Considering the quantity of recombinant product and the applicability of purification, the pET28-scygonadin expression system is a better choice to produce large quantities of recombinant scygonadin for commercial use in future. This is the first report on the heterologous expression of antimicrobial peptide scygonadin in E. coli.

A Closed-Tube Loop-Mediated Isothermal Amplification Assay for the Visual Detection of Staphylococcus aureus.

Food-borne diseases induced by Staphylococcus aureus contamination seriously affect human health and food safety. Therefore, a closed-tube loop-mediated isothermal amplification (LAMP) assay for the visual detection of S. aureus was developed in this study. Firstly, two pairs of outer and inner primers were designed targeting on a conserved fragment of gyrB gene in different S. aureus strains. Secondly, the weakly buffered gyrB-LAMP assays were optimized under various pH values and other conditions, followed by the visual evaluation of five pH-sensitive indicators, and the cresol-red was chosen as the best dye for the best visual performance. Thirdly, the cresol-red-based LAMP assay showed good sensitivity with the detection limit of 5.4 copies/µL for purified DNAs, and good specificity with no cross-reaction with other related species. The specificity of the amplified products was further confirmed by XbaI restriction enzyme digestion analysis. Finally, the cresol-red-based LAMP assay was validated by the clinical-dried fish samples inoculated with known numbers of S. aureus and further validated by 20 blind samples. To our knowledge, this is the first report of a closed-tube LAMP assay based on pH-sensitive indicators for the visual detection of the food-borne S. aureus by the gyrB gene.

Effect of asafoetida extract on growth and quality of Pleurotus ferulic.

Different concentrations of asafoetida extract were added to the medium of Pleurotus ferulic and the effects of the extract on growth of P. ferulic mycelium and fruiting bodies was observed. As the amount of asafoetida extract additive was increased, the growth of Pleurotus mycelium was faster, the time formation of buds was shorter and that yield of fruiting bodies was stimulated. However, overdosing of asafoetida extract hampered the growth of Pleurotus ferulic. The amino acid composition and volatile components in three kinds of pleurotus' were contrasted, including wild pleurotus (WP), cultivated pleurotus with asafoetida extract (CPAE) and cultivated pleurotus without asafoetida extract (CP). CPAE with 2.3 g/100 g asafoetida extract addition had the highest content of total amino acids, as well as essential amino acids. WP had a higher content of total amino acids and essential amino acids than CP. In addition, CPAE with 2.3 g/100 g had the highest score of protein content of pleurotus fruiting bodies, while WP had a higher score than CP. In the score of essential amino acid components of pleurotus fruiting bodies, CP had the highest score, while CPAE was higher than WP. Asafoetida extract influenced the volatile components of Pleurotus ferulic greatly, making the volatile components of cultivated pleurotus more similar to those of wild pleurotus (WP).

Effect of pulsed electric fields (PEF) on physico-chemical properties, ß-carotene and antioxidant activity of air-dried apricots.

Fresh apricots pre-treated by pulsed electric fields at different intensities [LPEF, 0.65 kV/cm, 100 Hz, 20 µs and total treatment time 30 s; HPEF1, 1.25 kV/cm, 100 Hz, 20 µs and total treatment time 30 s; HPEF2, 1.25 kV/cm, 100 Hz, 20 µs and total treatment time 60 s], along with controls [non-treated, non-treated and sulphite treated, and heat pre-treatment at 80 °C, for 10 min (HC)] and soaked in 0.2% sodium sulphite solution for 1 h and then were subject to hot air drying. The changes in drying rate, polyphenol oxidase, peroxidase, and ß-carotene contents as well as antioxidant activity and colour in pre-treatment and hot air-dried apricot samples were investigated. PEF and heat treatments increased the drying rate of apricots. PEF treatments had no effect on the PPO activity and decreased the POD activity (p < 0.05). HPEF2 treatment retained more ß-carotene, higher antioxidant activity and suffered less browning during processing. Overall, the results indicate that combining sulphite treatment with PEF produces dried apricots with more ß-carotene and antioxidant activity, and better colour.