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Aflatoxin B1 (AFB1) is a highly toxic mycotoxin, which causes severe acute or cumulative poisoning. Therefore, it is important to develop sensitive and selective detection methods for AFB1 for the safety of food and medicinal herbs. Herein, we have developed a "signal-on" electrochemical aptasensor based on the high specificity of the aptamer and hybridization chain reaction (HCR) biological amplification for AFB1 detection. In this work, thiol-modified complementary DNA (cDNA) immobilized on the surface of a gold electrode (GE) served as an initiator DNA. When AFB1 was present, it competed with the cDNA for binding to the aptamers, which resulted in the detaching of aptamers from the cDNA-aptamer duplexes. Then, the single-stranded cDNA acted as an initiator to trigger the HCR signal amplification. Therefore, long double-stranded DNA (dsDNA) products were produced, which could load large amounts of methylene blue (MB) molecules to generate a distinct electrochemical signal. Under the optimized conditions, the proposed electrochemical aptasensor achieved the ultrasensitive detection of AFB1 with a linear detection range of 0.01-100 pg mL

Rift Valley fever virus (RVFV) causes Rift Valley fever (RVF), resulting in morbidity and mortality in humans and ruminants. Evidence of transboundary outbreaks means that RVFV remains a threat to human health and livestock industries in countries that are free from the disease. To enhance surveillance capability, methods for detection of RVFV are required. The generation of reagents suitable for the detection of RVFV antigen in formalin-fixed, paraffin-embedded tissues from infected animals have been developed and are described herein. Recombinant nucleoprotein (rNP) was expressed in Escherichia coli and purified using immobilized metal ion affinity chromatography. Purified rNP was used as an immunogen to produce anti-NP polyclonal antisera in rabbits for use in detection of RVFV NP in experimentally infected animals by immunohistochemistry. Antisera raised in rabbits against rNP were able to recognize viral NP antigen in fixed infected Vero cell pellets and sheep liver. Therefore, the methods and reagents described herein are useful in assays for detection of RVFV infections in animals, for research and surveillance purposes.

Agar is modified by chemical methods to improve its functional properties and meet the increasing demand of the market. Some of the functional properties of agar are improved after chemical modification, while other properties are reduced, especially gel strength. This study aimed to comprehensively improve the functional properties of agar through acylation and crosslinking by reacting with maleic anhydride.

Chemical modification is often used to improve the gel properties of agar but inevitably weakens gel strength in practical applications. This study achieved a breakthrough in improving the gel properties of agar without reducing its gel strength through modification with succinic anhydride. Fourier transform infrared spectroscopy and carbon nuclear magnetic resonance analyses showed that succinic anhydride could be mono-succinylated, cross-linked, and desulfurized with agar. The transition from mono-succinylation to cross-linking of agar was achieved by attemperation. Interestingly, the gel transparency of mono-succinylated agar increased from 55% to 89%, but its gel strength remarkably decreased from 1073 g/cm

Remodelling of the extracellular matrix (ECM) by ECM metalloproteinases is increasingly being associated with regulation of immune cell function. ECM metalloproteinases, including Matrix Metalloproteinases (MMPs), A Disintegrin and Metalloproteinases (ADAMs) and ADAMs with Thombospondin-1 motifs (ADAMTS) play a vital role in pathogen defence and have been shown to influence migration of immune cells. This review provides a current summary of the role of ECM enzymes in immune cell migration and function and discusses opportunities and limitations for development of diagnostic and therapeutic strategies targeting metalloproteinase expression and activity in the context of infectious disease.

[This corrects the article DOI: 10.1371/journal.pbio.1002580.].

Difficulties associated with efficient delivery and targeting of miRNAs to cells is hampering the real world application of miRNA technology. This study utilized an influenza A-based delivery system to express miR-155 in order to knockdown SOCS1 mRNA. Using qPCR and dual luciferase technology we show that miR-155 delivery resulted in a significant increase in cellular miR-155 which facilitated a downregulation of SOCS1 gene expression and a functional increase in IL-6 and IFN-β cytokines.

Vaccination remains the most effective means by which influenza virus infection can be controlled. Traditionally vaccine production has focused on egg-based technology. Increasingly cell culture-based vaccine production is being used to improve preparedness. Cell cultured based production methods offer significant advantages over traditional egg-based strategies by allowing (i) rapid scale-up (ii) elimination of allergic egg components and (iii) circumvention of issues relating to egg-adaption. Our aim was to improve influenza vaccines and preparedness through the establishment of mammalian cell (Vero)-based vaccines using plasmid-based recombinant flu constructs expressing exogenous miRNA19 (flu-miR19), a regulator of key host anti-viral pathways that has previously been shown to modulate influenza virus replication. We successfully rescued recombinant influenza viruses expressing miR19 using reverse genetics; validated expression of miR19 and showed increased virus replication in vitro that corresponded with increased HA titres and matrix gene expression. Knockdown of miR19 host target genes was confirmed through qRT-PCR to validate the mechanism of action. This new influenza vaccine platform is scalable and can be merged with existing production technology to offer rapid clinical translation and improve vaccine quality and production times. In summary, silencing of anti-viral host genes using a flu-miR resulted in an increase of influenza vaccine and HA antigen yield. We now plan to further augment antigen yield by propagating these viruses in enhanced Vero cell vaccine substrates.

We used vertical growth phase (VGP) human VMM5 melanoma cells to ask whether the tumor microenvironment could induce matrix metalloproteinase-1 (MMP-1) in vivo, and whether this induction correlated with metastasis. We isolated two clones from parental VMM5 cells: a low MMP-1 producing clone (C4) and high producing clone (C9). When these clones were injected orthotopically (intradermally) into nude mice, both were equally tumorigenic and produced equivalent and abundant amounts of MMP-1. However, the tumors from the C4 clones displayed different growth kinetics and distinct profiles of gene expression from the C9 population. The C4 tumors, which had low MMP-1 levels in vitro, appeared to rely on growth factors and cytokines in the microenvironment to increase MMP-1 expression in vivo, while MMP-1 levels remained constant in the C9 tumors. C9 cells, but not C4 cells, grew as spheres in culture and expressed higher levels of JARID 1B, a marker associated with melanoma initiating cells. We conclude that VMM5 melanoma cells exhibit striking intra-tumor heterogeneity, and that the tumorigenicity of these clones is driven by different molecular pathways. Our data suggest that there are multiple mechanisms for melanoma progression within a tumor, which may require different therapeutic strategies.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl(-) currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.

Extracellular matrix (ECM) enzymes including the ADAMTS (A disintegrin-like and metalloproteinase with thrombospondin-1 motifs) family have been shown to regulate influenza-specific immunity. ADAMTS7, a largely uncharacterised ECM enzyme with broad tissue distribution has been associated with coronary heart disease and arthritis, however its role in viral immunity remains undefined. We assessed the contribution of ADAMTS7 to influenza-specific immunity using Adamts7−/− mice. Adamts7−/− mice intranasally infected with influenza virus showed increased weight loss and had higher viral loads when compared to WT controls. We also observed fewer influenza-specific CD8+ T cells and changes in cytokine production following primary and secondary infection suggesting ADAMTS7 expression is a key regulator of viral immunity.

Approximately one-quarter of people over the age of 65 are estimated to suffer some form of cognitive impairment, underscoring the need for effective cognitive-enhancing agents. Insulin-regulated aminopeptidase (IRAP) is potentially an innovative target for the development of cognitive enhancers, as its peptide inhibitors exhibit memory-enhancing effects in both normal and memory-impaired rodents. Using a homology model of the catalytic domain of IRAP and virtual screening, we have identified a class of nonpeptide, small-molecule inhibitors of IRAP. Structure-based computational development of an initial "hit" resulted in the identification of two divergent families of compounds. Subsequent medicinal chemistry performed on the highest affinity compound produced inhibitors with nanomolar affinities (K(i) 20-700 nM) for IRAP. In vivo efficacy of one of these inhibitors was demonstrated in rats with an acute dose (1 nmol in 1 microl) administered into the lateral ventricles, improving performance in both spatial working and recognition memory paradigms. We have identified a family of specific IRAP inhibitors that is biologically active which will be useful both in understanding the physiological role of IRAP and potentially in the development of clinically useful cognitive enhancers. Notably, this study also provides unequivocal proof of principal that inhibition of IRAP results in memory enhancement.

Insulin-regulated aminopeptidase, IRAP, is an abundant protein that was initially cloned from a rat epididymal fat pad cDNA library as a marker protein for specialized vesicles containing the insulin-responsive glucose transporter GLUT4, wherein it is thought to participate in the tethering and trafficking of GLUT4 vesicles. The same protein was independently cloned from human placental cDNA library as oxytocinase and is proposed to have a primary role in the regulation of circulating oxytocin (OXY) during the later stages of pregnancy. More recently, IRAP was identified as the specific binding site for angiotensin IV, and we propose that it mediates the memory-enhancing effects of the peptide. This protein appears to have multiple physiological roles that are tissue- and domain-specific; thus the protein can be specifically targeted for treating different clinical conditions.

We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the c ystic f ibrosis t ransmembrane conductance r egulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif , for C FTR i nhibitory f actor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl − ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in α/β hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe.

Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) accelerate spatial learning and facilitate memory retention and retrieval by binding competitively to the catalytic site of the enzyme and inhibiting its catalytic activity. IRAP belongs to the M1 family of Zn2+-dependent aminopeptidases characterized by a catalytic domain that contains two conserved motifs, the HEXXH(X)18E Zn2+-binding motif and the GXMEN exopeptidase motif. To elucidate the role of GXMEN in binding peptide substrates and competitive inhibitors, site-directed mutagenesis was performed on the motif. Non-conserved mutations of residues G428, A429 and N432 resulted in mutant enzymes with altered catalytic activity, as well as divergent changes in kinetic properties towards the synthetic substrate leucine β-naphthalamide. The affinities of the IRAP inhibitors angiotensin IV, Nle1-angiotensin IV, and LVV-hemorphin-7 were selectively decreased. Substrate degradation studies using the in vitro substrates vasopressin and Leu-enkephalin showed that replacement of G428 by either D, E or Q selectively abolished the catalysis of Leu-enkephalin, while [A429G]IRAP and [N432A]IRAP mutants were incapable of cleaving both substrates. These mutational studies indicate that G428, A429 and N432 are important for binding of both peptide substrates and inhibitors, and confirm previous results demonstrating that peptide IRAP inhibitors competitively bind to its catalytic site.

Insulin-regulated aminopeptidase (IRAP), a marker of glucose transporter 4 (GLUT4) storage vesicles (GSVs), is the only protein known to traffic with GLUT4. In the basal state, GSVs are sequestered from the constitutively recycling endosomal system to an insulin-responsive, intracellular pool. Insulin induces a rapid translocation of GSVs to the cell surface from this pool, resulting in the incorporation of IRAP and GLUT4 into the plasma membrane. We sought to identify proteins that interact with IRAP to further understand this GSV trafficking process. This study describes our identification of a novel interaction between the amino terminus of IRAP and the Akt substrate, AS160 (Akt substrate of 160 kDa). The validity of this interaction was confirmed by coimmunoprecipitation of both overexpressed and endogenous proteins. Moreover, confocal microscopy demonstrated colocalization of these proteins. In addition, we demonstrate that the IRAP-binding domain of AS160 falls within its second phosphotyrosine-binding domain and the interaction is not regulated by AS160 phosphorylation. We hypothesize that AS160 is localized to GLUT4-containing vesicles via its interaction with IRAP where it inhibits the activity of Rab substrates in its vicinity, effectively tethering the vesicles intracellularly.

Receptor activity-modifying proteins (RAMPs) interact with and modify the behavior of the calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR). We have examined the contribution of the short intracellular C terminus, using constructs that delete the last eight amino acids of each RAMP. C-Terminal deletion of individual RAMPs had little effect on the signaling profile induced when complexed with CLR in COS-7 or human embryonic kidney (HEK)293 cells. Likewise, confocal microscopy revealed each of the mutant RAMPs translocated hemagglutinin-tagged CLR to the cell surface. In contrast, a pronounced effect of RAMP C-terminal truncation was seen for RAMP/CTRa complexes, studied in COS-7 cells, with significant attenuation of amylin receptor phenotype induction that was stronger for RAMP1 and -2 than RAMP3. The loss of amylin binding upon C-terminal deletion could be partially recovered with overexpression of Galpha(s), suggesting an impact of the RAMP C terminus on coupling of G proteins to the receptor complex. In HEK293 cells the c-Myc-RAMP1 C-terminal deletion mutant showed high receptor-independent cell surface expression; however, this construct showed low cell surface expression when expressed alone in COS-7 cells, indicating interaction of RAMPs with other cellular components via the C terminus. This mutant also had reduced cell surface expression when coexpressed with CTR. Thus, this study reveals important functionality of the RAMP C-terminal domain and identifies key differences in the role of the RAMP C terminus for CTR versus CLR-based receptors.

Background: The secretory leukocyte protease inhibitor (SLPI) represents a multifunctional protein with mucosa-protective features. Helicobacter pylori and the usage of low-dose aspirin are two independent risk factors for the development of gastrointestinal diseases. Therefore, the effect of low-dose aspirin on gastrointestinal SLPI expression was analyzed in the context of H. pylori infection. Material and Methods: The study included 20 volunteers (H. pylori positive and negative: n = 10) who received 2 × 50 mg aspirin/day for 7 days. H. pylori-positive subjects underwent eradication therapy and repeated the protocol. Gastroduodenoscopy was performed at day 0, 1, 3, and 7, and biopsies were obtained each from antrum, corpus, and duodenal bulb. SLPI expression was determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Results: A reduction of antral SLPI levels, ranging between 582 (day 0) and 941 pg/10 µg protein (day 7), was determined in H. pylori-positive compared to H. pylori-negative and -eradicated subjects (1600–2050 pg/10 µg protein, ANOVA: p = .001–.045). No differences concerning aspirin were observed within the groups. SLPI levels in corpus and duodenal mucosa were neither affected by H. pylori nor low-dose aspirin. There was an inverse correlation between SLPI and H. pylori-induced inflammation (activity: r = −0.575, −0.69 to −0.43, p

Highly pathogenic avian influenza virus infection is associated with severe mortality in both humans and poultry. The mechanisms of disease pathogenesis and immunity are poorly understood although recent evidence suggests that cytokine/chemokine dysregulation contributes to disease severity following H5N1 infection. Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased reactive oxygen species production, cytokine expression, and acute lung injury. Proinflammatory stimuli are known to induce intracellular reactive oxygen species by activating NADPH oxidase activity. We therefore hypothesized that inhibition of this activity would restore host cytokine homeostasis following avian influenza virus infection. A panel of airway epithelial and immune cells from mammalian and avian species were infected with A/Puerto Rico/8/1934 H1N1 virus, low-pathogenicity avian influenza H5N3 virus (A/duck/Victoria/0305-2/2012), highly pathogenic avian influenza H5N1 virus (A/chicken/Vietnam/0008/2004), or low-pathogenicity avian influenza H7N9 virus (A/Anhui/1/2013). Quantitative real-time reverse transcriptase PCR showed that H5N1 and H7N9 viruses significantly stimulated cytokine (interleukin-6, beta interferon, CXCL10, and CCL5) production. Among the influenza-induced cytokines, CCL5 was identified as a potential marker for overactive immunity. Apocynin, a Nox2 inhibitor, inhibited influenza-induced cytokines and reactive oxygen species production, although viral replication was not significantly altered in vitro . Interestingly, apocynin treatment significantly increased influenza virus-induced mRNA and protein expression of SOCS1 and SOCS3, enhancing negative regulation of cytokine signaling. These findings suggest that apocynin or its derivatives (targeting host responses) could be used in combination with antiviral strategies (targeting viruses) as therapeutic agents to ameliorate disease severity in susceptible species. IMPORTANCE Highly pathogenic avian influenza virus infection causes severe morbidity and mortality in both humans and poultry. Wide-spread antiviral resistance necessitates the need for the development of additional novel therapeutic measures to modulate overactive host immune responses after infection. Disease severity following avian influenza virus infection can be attributed in part to hyperinduction of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species. Our study shows that highly pathogenic avian influenza H5N1 virus and low-pathogenicity avian influenza H7N9 virus (both associated with human fatalities) promote inactivation of FoxO3 and downregulation of the TAM receptor tyrosine kinase, Tyro3, leading to augmentation of the inflammatory cytokine response. Inhibition of influenza-induced reactive oxygen species with apocynin activated FoxO3 and stimulated SOCS1 and SOCS3 proteins, restoring cytokine homeostasis. We conclude that modulation of host immune responses with antioxidant and/or anti-inflammatory agents in combination with antiviral therapy may have important therapeutic benefits.

The angiotensin AT4 receptor was originally defined as the specific, high-affinity binding site for the hexapeptide angiotensin IV (Ang IV). Subsequently, the peptide LVV-hemorphin 7 was also demonstrated to be a bioactive ligand of the AT4 receptor. Central administration of Ang IV, its analogues or LVV-hemorphin 7 markedly enhance learning and memory in normal rodents and reverse memory deficits observed in animal models of amnesia. The AT4 receptor has a broad distribution and is found in a range of tissues, including the adrenal gland, kidney, lung and heart. In the kidney Ang IV increases renal cortical blood flow and decreases Na+ transport in isolated renal proximal tubules. The AT4 receptor has recently been identified as the transmembrane enzyme, insulin-regulated membrane aminopeptidase (IRAP). IRAP is a type II integral membrane spanning protein belonging to the M1 family of aminopeptidases and is predominantly found in GLUT4 vesicles in insulin-responsive cells. Three hypotheses for the memory-potentiating effects of the AT4 receptor/IRAP ligands, Ang IV and LVV-hemorphin 7, are proposed: (i) acting as potent inhibitors of IRAP, they may prolong the action of endogenous promnestic peptides; (ii) they may modulate glucose uptake by modulating trafficking of GLUT4; (iii) IRAP may act as a receptor, transducing the signal initiated by ligand binding to its C-terminal domain to the intracellular domain that interacts with several cytoplasmic proteins.

In mammals the M1 aminopeptidase family consists of nine different proteins, five of which are integral membrane proteins. The aminopeptidases are defined by two motifs in the catalytic domain; a zinc binding motif HEXXH-(X18)-E and an exopeptidase motif GXMEN. Aminopeptidases of this family are able to cleave a broad range of peptides down to only to a single peptide. This ability to either generate or degrade active peptide hormones is the focus of this review. In addition to their capacity to degrade a range of peptides a number of these aminopeptidases have novel functions that impact on cell signalling and will be discussed.

Angiotensin IV (Ang IV) exerts profound effects on memory and learning, a phenomenon ascribed to its binding to a specific AT4 receptor. However the AT4 receptor has recently been identified as the insulin-regulated aminopeptidase (IRAP). In this study, we demonstrate that AT4 receptor ligands, including Ang IV, Nle1-Ang IV, divalinal-Ang IV, and the structurally unrelated LVV-hemorphin-7, are all potent inhibitors of IRAP catalytic activity, as assessed by cleavage of leu-β-naphthylamide by recombinant human IRAP. Both Ang IV and divalinal–Ang IV display competitive kinetics, indicating that AT4 ligands mediate their effects by binding to the catalytic site of IRAP. The AT4 ligands also displaced [125I]-Nle1-Ang IV or [125I]-divalinal1-Ang IV from IRAP-HEK293T membranes with high affinity, which was up to 200-fold greater than in the catalytic assay; this difference was not consistent among the peptides, and could not be ascribed to ligand degradation. Although some AT4 ligands were subject to minor cleavage by HEK293T membranes, none were substrates for IRAP. Of a range of peptides tested, only vasopressin, oxytocin, and met-enkephalin were rapidly cleaved by IRAP. We propose that the physiological effects of AT4 ligands result, in part, from inhibition of IRAP cleavage of neuropeptides involved in memory processing.

Members of the ADAMTS (A disintegrin-like and metalloproteinase with thrombospondin-1 motifs) metalloproteinase family remodel the extracellular matrix (ECM). In this current study we demonstrate that Adamts5−/− mice infected with X31 influenza virus (104pfu/mouse) show enhanced weight loss, have higher virus titres and perturbed T cell immunity when compared to WT counterparts. These responses correlate with the accumulation of a key ADAMTS5 substrate, versican, in the MLN of Adamts5−/− mice and underpin disruption of T cell migration as evidenced through the use of both in vitro and in vivo models. Our research emphasizes the importance of ADAMTS5 activity in the control of influenza virus infection and highlights the potential for development of ADAMTS5-based therapeutic strategies to reduce overall morbidity and mortality.

Toll-like receptor (TLRs) is a type of pattern-recognition receptor that recognizes pathogen-associated molecular patterns, the important mediator of innate immunity. The aim of this study was to examine the anti-inflammatory effect of TLR2 monoclonal antibody (TLR2 mAb) on concanavalin A (ConA) induced-hepatitis. Our in vitro findings indicated that the TLR2 monoclonal antibody developed by us was able to neutralize the activation of peripherial blood mononucleated cells (PBMC) induced by ConA. Furthermore, pretreatment of mice with TLR2 antibody exerted liver protection against ConA. Compared with the isotype IgG group, the TLR2-antibody-treated group demonstrated less mortality with concomitant decreased serum level of Ast and Alt. The detailed mechanisms underlying the protection effect induced by TLR2 antibody was due to inhibition of infiltration of lymphocytes in liver induced by ConA as well as the expression of pro-inflammatory factors, such as TNF-α, IFN-γ, IL-6. Taken together, our findings demonstrated that TLR2 mAb pretreatment protected liver against ConA-induced hepatitis in mice, suggesting that TLR2 mAb is a potential candidate for therapy of acute hepatitis.

Reverse genetics systems allow artificial generation of non-segmented and segmented negative-sense RNA viruses, like influenza viruses, entirely from cloned cDNA. Since the introduction of reverse genetics systems over a decade ago, the ability to generate ‘designer’ influenza viruses in the laboratory has advanced both basic and applied research, providing a powerful tool to investigate and characterise host–pathogen interactions and advance the development of novel therapeutic strategies. The list of applications for reverse genetics has expanded vastly in recent years. In this review, we discuss the development and implications of this technique, including the recent controversy surrounding the generation of a transmissible H5N1 influenza virus. We will focus on research involving the identification of viral protein function, development of live-attenuated influenza virus vaccines, host–pathogen interactions, immunity and the generation of recombinant influenza virus vaccine vectors for the prevention and treatment of infectious diseases and cancer.

The extracellular matrix (ECM) provides physical scaffolding for cellular constituents and initiates biochemical and biomechanical cues that are required for physiological activity of living tissues. The ECM enzyme ADAMTS5, a member of the ADAMTS (A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs) protein family, cleaves large proteoglycans such as aggrecan, leading to the destruction of cartilage and osteoarthritis. However, its contribution to viral pathogenesis and immunity is currently undefined. Here, we use a combination of in vitro and in vivo models to show that ADAMTS5 enzymatic activity plays a key role in the development of influenza-specific immunity. Influenza virus infection of Adamts5-/- mice resulted in delayed virus clearance, compromised T cell migration and immunity and accumulation of versican, an ADAMTS5 proteoglycan substrate. Our research emphasises the importance of ADAMTS5 expression in the control of influenza virus infection and highlights the potential for development of ADAMTS5-based therapeutic strategies to reduce morbidity and mortality., The extracellular matrix enzyme ADAMTS5 enhances the clearance of viruses by facilitating migration of T lymphocytes to the periphery following influenza virus infection., Author Summary Movement of immune cells is critical for effective clearance of pathogens. The response to influenza virus infection requires immune cell trafficking between the lung, mediastinal lymph node and other peripheral lymphoid organs such as the spleen. We set out to assess the contribution of a specific extracellular matrix enzyme, ADAMTS5, to migration of lymphocytes and overall pathogenesis following infection. In our studies, we demonstrate that mice lacking Adamts5 have fewer influenza-specific lymphocytes in the lung and spleen following infection. These observations correlated with an accumulation of influenza-specific lymphocytes in the mediastinal lymph node and increased virus titres. This work suggests that ADAMTS5 is necessary for immune cell migration to the periphery, where lymphocyte function is required to fight infection.

Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8(+) T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.

Current influenza vaccines offer considerable protection for the majority of the population. However, vaccine efficacy amongst high risk populations, such as the elderly, is variable. Novel approaches are therefore required to improve vaccine efficiency in these susceptible populations. MicroRNAs (miRNAs) play important roles in the regulation of immune responses by modulating gene expression. Our work describes the generation and delivery of engineered influenza viruses with the ability to express defined immunomodulatory miRNAs. MicroRNA-150 (miR-150) has been shown to be involved in the development of T cell and B cell. Furthermore, evidence suggests that circulating miR-150 can be actively transported to target cells where it can influence gene expression. In this study, we first investigated the ability of recombinant influenza viruses to deliver immunomodulatory miR-150, and alter cytokine production in vitro. miR-150 was inserted into an artificial intron between the NS1 and NEP genes of the PR8 H1N1(PR8-150) and X31 H3N2 (X31-150) influenza A viruses and was rescued using reverse genetics. Mature miR-150 expression was confirmed using qPCR and by knockdown of its target gene, c-Myb, using a dual-luciferase assay. Moreover, cytokine mRNA expression levels (IL-6, IFN-β, CXCL10 and CCL5) were enhanced following infection with PR8-150 in A549 cells. Mice infected intranasally with recombinant X31-150 virus showed significantly higher numbers of neutrophils, macrophages and influenza specific CD8+ T cells post infection compared to X31 wild type controls. These results demonstrate that recombinant influenza viruses can be used to deliver functional miRNAs with the ability to modulate immunity both in vitro and in vivo.

Members of the ADAMTS (A disintegrin-like and metalloproteinase with thrombospondin-1 motifs) metalloproteinase family remodel the extracellular matrix (ECM) to facilitate immune cell migration and cell adhesion. In this current study, we infected Adamts5−/− mice with X31 influenza virus (104pfu/mouse)and measured weight loss, viraemia and T cell immunity following acute infection. Adamts5−/− mice showed greater weight loss and increased viraemia when compared to WT counterparts. Furthermore, we observed reduced NP366 and PA224 influenza specific CD8+ T cells in the spleen and lungs of Adamts5−/− mice with a corresponding accumulation of these T cell specificities in the mediastinal lymph node (MLN) 10 days post infection. Critically, these responses correlated with increased expression of a key ADAMTS5 substrate; versican, in the MLNs of Adamts5−/− mice. We therefore hypothesize that versican accumulation in the MLNs of Adamts5−/− mice results in T cell accumulation and that ADAMTS5 is a key enzyme involved in facilitating T cell migration from the MLN to the periphery following acute influenza virus infection.

To set up a method of amplification for the whole CagA gene of Helicobacter pylori and its fingerprinting by restriction fragment length polymorphism (RFLP), nested PCR was employed in combination with TD-PCR to amplify the gene and EcoRI and Hind III were used to generate the RFLP fingerprinting. Target DNA fragments from 13 of 20 samples were successfully amplified and the relevant RFLP fingerprintings were obtained. It is concluded that the method can be used to amplify the whole CagA gene and CagA gene has apparent diversity of RFLP profile.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD), pneumonia, cystic fibrosis (CF), and bronchiectasis. Cif (PA2934), a bacterial toxin secreted in outer membrane vesicles (OMV) by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens., National Institutes of Health (U.S.) (NIH grants R01-HL074175), National Institutes of Health (U.S.) (R01-DK/HL-45881), National Institutes of Health (U.S.) (5P20RR018787-08), National Institutes of Health (U.S.) (R01-AI083256), Cystic Fibrosis Foundation (CF RDP), Cystic Fibrosis Foundation (BOMBER08F0)

Inhibitors of insulin-regulated aminopeptidase (IRAP) improve memory and are being developed as a novel treatment for memory loss. In this study, the binding of a class of these inhibitors to human IRAP was investigated using molecular docking and site-directed mutagenesis. Four benzopyran-based IRAP inhibitors with different affinities were docked into a homology model of the catalytic site of IRAP. Two 4-pyridinyl derivatives orient with the benzopyran oxygen interacting with the Zn(2+) ion and a direct parallel ring-stack interaction between the benzopyran rings and Phe544. In contrast, the two 4-quinolinyl derivatives orient in a different manner, interacting with the Zn(2+) ion via the quinoline nitrogen, and Phe544 contributes an edge-face hydrophobic stacking point with the benzopyran moiety. Mutagenic replacement of Phe544 with alanine, isoleucine, or valine resulted in either complete loss of catalytic activity or altered hydrolysis velocity that was substrate-dependent. Phe544 is also important for inhibitor binding, because these mutations altered the K(i) in some cases, and docking of the inhibitors into the corresponding Phe544 mutant models revealed how the interaction might be disturbed. These findings demonstrate a key role of Phe544 in the binding of the benzopyran IRAP inhibitors and for optimal positioning of enzyme substrates during catalysis.

P-glycoprotein (Pgp), a member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily, is a major drug efflux pump expressed in normal tissues, and is overexpressed in many human cancers. Overexpression of Pgp results in reduced intracellular drug concentration and cytotoxicity of chemotherapeutic drugs and is thought to contribute to multidrug resistance of cancer cells. The involvement of Pgp in clinical drug resistance has led to a search for molecules that block Pgp transporter activity to improve the efficacy and pharmacokinetics of therapeutic agents. We have recently identified and characterized a secreted toxin from Pseudomonas aeruginosa, designated cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif). Cif reduces the apical membrane abundance of CFTR, also an ABC transporter, and inhibits the CFTR-mediated chloride ion secretion by human airway and kidney epithelial cells. We report presently that Cif also inhibits the apical membrane abundance of Pgp in kidney, airway, and intestinal epithelial cells but has no effect on plasma membrane abundance of multidrug resistance protein 1 or 2. Cif increased the drug sensitivity to doxorubicin in kidney cells expressing Pgp by 10-fold and increased the cellular accumulation of daunorubicin by 2-fold. Thus our studies show that Cif increases the sensitivity of Pgp-overexpressing cells to doxorubicin, consistent with the hypothesis that Cif affects Pgp functional expression. These results suggest that Cif may be useful to develop a new class of specific inhibitors of Pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs, and at improving the bioavailability of Pgp transport substrates.

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing ΔF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and ΔF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on ΔF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing ΔF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The ΔF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

The angiotensin AT(4) receptor was originally defined as the specific, high affinity binding site for the hexapeptide angiotensin IV (Ang IV). Subsequently, the peptide LVV-hemorphin 7 was also demonstrated to be a bioactive ligand of the AT(4) receptor. Central administration of Ang IV or LVV-hemorphin 7 (LVV-H7) markedly enhances learning and memory in normal rodents and reverse memory deficits observed in animal models of amnesia. The high affinity binding site has a broad distribution in the brain including areas such as the hippocampus that are involved in memory processing. The high affinity Ang IV binding site (AT(4) receptor) has been identified as the transmembrane enzyme, insulin-regulated membrane aminopeptidase (IRAP). Insulin-regulated aminopeptidase is a type II integral membrane spanning protein belonging to the M1 family of aminopeptidases and in insulin-responsive cells colocalizes with GLUT4 in specific intra-cellular vesicles. Both Ang IV and LVV-H7 are competitive inhibitors of IRAP catalytic activity and are not substrates of the enzyme.

Although insulin-regulated aminopeptidase (IRAP) was first described as a marker protein for specialized vesicles containing the insulin-responsive glucose transporter, GLUT4, the protein has subsequently been shown to be identical to oxytocinase or the AT4 receptor. In insulin responsive tissues, IRAP is almost exclusively co-localized with GLUT4, being retained in intracellular compartments in the basal state or translocating with GLUT4 to the plasma membrane under insulin stimulation. The function of IRAP in these tissues has not been elucidated — the protein is thought to be involved in the tethering of GLUT4 vesicles to intracellular compartments. In the placenta, IRAP was isolated as the enzyme that degrades oxytocin and is therefore thought to play a role in the prevention of premature labour and maintenance of an adequate blood flow to the uterus. Potentially the most exciting physiological role attributed to IRAP is the involvement of the enzyme in memory processing. AT4 ligands, upon binding to the catalytic site of IRAP, enhance spatial learning, facilitate memory retention and retrieval and reverse amnesia. We postulate that the AT4 ligands act via either one of these mechanisms: (1) The peptides bind to the catalytic site of IRAP and inhibit its enzymatic activity thereby prolonging the half-life of its neuropeptide substrates with memory-enhancing properties or (2) Upon binding to IRAP, the AT4 ligands regulate the level of GLUT4 expressed at the cell surface resulting in an increase in glucose uptake into neurones.

Angiotensin IV (Ang IV) exerts profound effects on memory and learning, a phenomenon ascribed to its binding to a specific AT4 receptor. However the AT4 receptor has recently been identified as the insulin-regulated aminopeptidase (IRAP). In this study, we demonstrate that AT4 receptor ligands, including Ang IV, Nle1-Ang IV, divalinal-Ang IV, and the structurally unrelated LVV-hemorphin-7, are all potent inhibitors of IRAP catalytic activity, as assessed by cleavage of leu-beta-naphthylamide by recombinant human IRAP. Both Ang IV and divalinal-Ang IV display competitive kinetics, indicating that AT4 ligands mediate their effects by binding to the catalytic site of IRAP. The AT4 ligands also displaced [125I]-Nle1-Ang IV or [125I]-divalinal1-Ang IV from IRAP-HEK293T membranes with high affinity, which was up to 200-fold greater than in the catalytic assay; this difference was not consistent among the peptides, and could not be ascribed to ligand degradation. Although some AT4 ligands were subject to minor cleavage by HEK293T membranes, none were substrates for IRAP. Of a range of peptides tested, only vasopressin, oxytocin, and met-enkephalin were rapidly cleaved by IRAP. We propose that the physiological effects of AT4 ligands result, in part, from inhibition of IRAP cleavage of neuropeptides involved in memory processing.

Influenza A virus infection causes acute inflammation resulting in increased reactive oxygen species (ROS) production and lung injury. ROS are produced following activation of the NADPH oxidase enzyme complex. Apocynin, an inhibitor of NADPH oxidase complex formation has been shown to reduce the accumulation of inflammatory cells in the lungs of influenza-infected mice. As hypercytokinemia is thought to contribute to severe disease pathogenesis of both H5N1 and H7N9 infection in humans, we hypothesized that apocynin could reduce virus-induced cytokine/chemokine production. Human lung epithelial and chicken cell lines were infected with low pathogenic avian influenza H7N9 (A/Anhui/1/2013) and HPAI H5N1 (A/chicken/Vietnam/0008/2004) viruses in the absence or presence of apocynin. Quantitative real-time PCR demonstrated that apocynin inhibited influenza-induced cytokines/chemokines and ROS production following infection. Interestingly, addition of apocynin to in vitro cultures significantly increased SOCS1 and SOCS3 mRNA and protein expression, contributing to the negative regulation of overall cytokine expression. We suggest that intervention strategies targeting both the virus (current therapeutics) and the host e.g. apocynin (future therapeutics) could be used to ameliorate disease in infected individuals, reducing hypercytokinemia and pathology and allowing full development of adaptive immune responses.