Stool Gluten Peptide Detection Is Superior to Urinary Analysis, Coeliac Serology, Dietary Adherence Scores and Symptoms in the Detection of Intermittent Gluten Exposure in Coeliac Disease: A Randomised, Placebo-Controlled, Low-Dose Gluten Challenge Study.

Monitoring adherence to a gluten-free diet is an important goal of coeliac disease management. Urine and stool gluten immunogenic peptide (GIP) assays provide an objective readout of gluten ingestion, with the former favoured due to its convenience and acceptability. This study assessed stool GIP excretion after low-dose gluten challenge designed to mimic accidental gluten exposure. A total of 52 coeliac participants undertook a randomised, double-blind gluten (50-1000 mg) or placebo challenge. Stool and urinary GIP, serology, dietary adherence and symptoms were assessed. Stool GIP was 100% sensitive for gluten intake ≥250 mg and 71% for 50 mg. Peak GIP detection was 12-36 h after gluten exposure. The mean stool GIP after 1000 mg gluten ingestion remained above the limit of quantification for 5 days. Urine GIP assessment had poor sensitivity for GIP excretion compared to stool. Serology, dietary adherence score and symptoms did not correlate with gluten excretion during lead-in. We conclude that stool GIP detection is highly sensitive, with levels related to gluten dose and time from ingestion. Weekly or bi-weekly testing will detect low-level exposure more effectively than urine GIP assessments or traditional methods. In this seronegative, apparently well-treated cohort, a high frequency of baseline-positive GIP suggests ongoing gluten exposure, but the assessment of patient behaviour and assay specificity is needed.

Commercial fish ELISA kits have a limited capacity to detect different fish species and their products.

BACKGROUND: Fish is a major food and allergen source, requiring safety declarations on packages. Enzyme-linked immunosorbent assays (ELISAs) are often used to ensure that the product meets the required standards with regard to the presence of allergens. Over 1000 different fish species are traded and consumed worldwide, and they are increasingly provided by aquaculture. Up to 3% of the general population is at risk of sometimes fatal allergic reactions to fish, requiring strict avoidance of this commodity. The aim of this study is to evaluate the capacity of three commercially available ELISA tests to detect a wide variety of bony and cartilaginous fish and their products, which is essential to ensure reliable and safe food labeling. RESULTS: The detection rates for 57 bony fish ranged from 26% to 61%. Common European and North American species, including carp, cod, and salmon species, demonstrated a higher detection rate than those from the Asia-Pacific region, including pangasius and several mackerel and tuna species. Among the 17 canned bony fish products, only 65% to 86% were detected, with tuna showing the lowest rate. None of the cartilaginous fish (n = 9), other vertebrates (n = 8), or shellfish (n = 5) were detected. CONCLUSIONS: We demonstrated that three commercial fish ELISA kits had a limited capacity to detect fish and their products. The complexity of fish as a protein source that is increasingly utilized means that there is an urgent need for improved detection methods. This is crucial for the food industry to provide safe seafood products and comply with international legislation. © 2020 Society of Chemical Industry.

Undeclared allergens in imported packaged food for retail in Australia.

The Australia New Zealand Food Standards Code (the Code) requires a declaration of the presence of 11 different allergens made through the label on a food product. Most food recalls in Australia are now due to undeclared allergens . This survey determined the extent of undeclared allergens in imported food products on the Asian retail market in Australia. A total of 50 imported packaged foods were selectively purchased from local Asian grocery retail stores in Melbourne and the presence of undeclared gluten, milk, peanut and egg determined. Analysis was performed using commercial enzyme-linked immunosorbent assay (ELISA) (R-Biopharm). Thirty-seven undeclared allergens (gluten n = 12, milk n = 12, peanut n = 6, and egg n = 7) were detected in 23 of the 50 products analysed (46%), with 18% containing multiple undeclared allergens. The high number of undeclared allergens is alarming and in line with the increasing number of food recalls and anaphylaxis recorded in Australia.

Lupine allergen detecting capability and cross-reactivity of related legumes by ELISA.

Lupine belongs to the genus Lupinus and includes three species commonly consumed by humans. The Lupinus genus is closely related to other legumes, such as peanuts, soya, chickpeas, peas, lentils and beans. However, the consumption of lupine (and related legumes) can cause severe allergenic reactions. Therefore, reliable analytical detection methods are required for the analysis of food samples. In this study three commercially available ELISA test kits were analyzed for the detection capability of three common lupine species, as well as cross-reactivity to related legumes. All three ELISA test kits could detect the lupine species, though with different sensitivities. Cross-reactivity varied for the ELISA test kits and all showed some cross-reactivity to related legume samples analyzed.

Food Allergen Management in Australia.

Food allergies are increasing globally, including numbers of allergens, the sensitization rate, and the prevalence rate. To protect food-allergic individuals in the community, food allergies need to be appropriately managed. This paper describes current Australian food allergen management practices. In Australia, the prevalence of food allergies, the anaphylaxis rate, and the fatal anaphylaxis rate are among the highest in the world. Interagency and stakeholder collaboration is facilitated and enhanced as Australia moves through past, current, and ongoing food allergen challenges. As a result, Australia has been a global leader in regulating the labeling of common allergens in packaged foods and their disclosure in foods not required to bear a label. Moreover, the food industry in Australia and New Zealand has developed a unique food allergen risk management tool, the Voluntary Incidental Trace Allergen Labelling program, which is managed by the Allergen Bureau. This paper summarizes insights and information provided by the major stakeholders involved to protect food-allergic consumers from any allergic reaction. Stakeholders include government; consumer protection, regulation, and enforcement agencies; the food industry; and food allergen testing and food allergen/allergy research bodies in Australia. The ongoing goal of all stakeholders in food allergen management in Australia is to promote best practice food allergen management procedures and provide a wide choice of foods, while enabling allergic consumers to manage their food allergies and reduce the risk of an allergic reaction.

Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins.

The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.

Next generation of food allergen quantification using mass spectrometric systems.

Food allergies are increasing worldwide and becoming a public health concern. Food legislation requires detailed declarations of potential allergens in food products and therefore an increased capability to analyze for the presence of food allergens. Currently, antibody-based methods are mainly utilized to quantify allergens; however, these methods have several disadvantages. Recently, mass spectrometry (MS) techniques have been developed and applied to food allergen analysis. At present, 46 allergens from 11 different food sources have been characterized using different MS approaches and some specific signature peptides have been published. However, quantification of allergens using MS is not routinely employed. This review compares the different aspects of food allergen quantification using advanced MS techniques including multiple reaction monitoring. The latter provides low limits of quantification for multiple allergens in simple or complex food matrices, while being robust and reproducible. This review provides an overview of current approaches to analyze food allergens, with specific focus on MS systems and applications.

A multi-laboratory evaluation of a clinically-validated incurred quality control material for analysis of allergens in food.

A dessert matrix previously used for diagnosis of food allergies was incurred with pasteurised egg white or skimmed milk powder at 3, 6, 15 and 30 mg allergen protein per kg of dessert matrix and evaluated as a quality control material for allergen analysis in a multi-laboratory trial. Analysis was performed by immunoassay using five kits each for egg and milk (based on casein) and six 'other' milk kits (five based on ß-lactoglobulin and one total milk). All kits detected allergen protein at the 3 mg kg(-1) level. Based on ISO criteria only one egg kit accurately determined egg protein at 3 mg kg(-1) (p=0.62) and one milk (casein) kit accurately determined milk at 6 (p=0.54) and 15 mg kg(-1) (p=0.83), against the target value. The milk "other" kits performed least well of all the kits assessed, giving the least precise analyses. The incurred dessert material had the characteristics required for a quality control material for allergen analysis.

Structure-guided design affirms inhibitors of hepatitis C virus p7 as a viable class of antivirals targeting virion release.

UNLABELLED: Current interferon-based therapy for hepatitis C virus (HCV) infection is inadequate, prompting a shift toward combinations of direct-acting antivirals (DAA) with the first protease-targeted drugs licensed in 2012. Many compounds are in the pipeline yet primarily target only three viral proteins, namely, NS3/4A protease, NS5B polymerase, and NS5A. With concerns growing over resistance, broadening the repertoire for DAA targets is a major priority. Here we describe the complete structure of the HCV p7 protein as a monomeric hairpin, solved using a novel combination of chemical shift and nuclear Overhauser effect (NOE)-based methods. This represents atomic resolution information for a full-length virus-coded ion channel, or "viroporin," whose essential functions represent a clinically proven class of antiviral target exploited previously for influenza A virus therapy. Specific drug-protein interactions validate an allosteric site on the channel periphery and its relevance is demonstrated by the selection of novel, structurally diverse inhibitory small molecules with nanomolar potency in culture. Hit compounds represent a 10,000-fold improvement over prototypes, suppress rimantadine resistance polymorphisms at submicromolar concentrations, and show activity against other HCV genotypes. CONCLUSION: This proof-of-principle that structure-guided design can lead to drug-like molecules affirms p7 as a much-needed new target in the burgeoning era of HCV DAA.

Invasive mammals and habitat modification interact to generate unforeseen outcomes for indigenous fauna.

Biotic invasions and habitat modification are two drivers of global change predicted to have detrimental impacts on the persistence of indigenous biota worldwide. Few studies have investigated how they operate synergistically to alter trophic interactions among indigenous and nonindigenous species in invaded ecosystems. We experimentally manipulated a suite of interacting invasive mammals, including top predators (cat Felis catus, ferret Mustela furo, stoat M. erminea), herbivores (rabbit Oryctolagus cuniculus, hare Lepus europaeus), and an insectivore (hedgehog Erinaceus europaeus occidentalis), and measured their effects on indigenous lizards and invertebrates and on an invasive mesopredator (house mouse Mus musculus). The work was carried out in a grassland/shrubland ecosystem that had been subjected to two types of habitat modification (widespread introduction of high-seed-producing pasture species, and areas of land use intensification by fertilization and livestock grazing). We also quantified food productivity for indigenous and invasive fauna by measuring pasture biomass, as well as seed and fruit production by grasses and shrubs. Indigenous fauna did not always increase following top-predator suppression: lizards increased on one of two sites; invertebrates did not increase on either site. Mesopredator release of mice was evident at the site where lizards did not increase, suggesting negative effects of mice on lizard populations. High mouse abundance occurred only on the predator-suppression site with regular production of pasture seed, indicating that this food resource was the main driver of mouse populations. Removal of herbivores increased pasture and seed production, which further enhanced ecological release of mice, particularly where pasture swards were overtopped by shrubs. An effect of landscape supplementation was also evident where nearby fertilized pastures boosted rabbit numbers and the associated top predators. Other studies have shown that both suppression of invasive predators and retiring land from grazing can benefit indigenous species, but our results suggest that the ensuing vegetation changes and complex interactions among invasive species can block recovery of indigenous fauna vulnerable to mesopredators. Top-down and bottom-up ecological release of mesopredators and landscape supplementation of top predators are key processes to consider when managing invaded communities in complex landscapes.

Inhibition of hepatitis C virus p7 membrane channels in a liposome-based assay system.

Chemotherapy for patients chronically infected with hepatitis C virus (HCV) is ineffective in over 50% of cases, generating a high demand for new drug targets. The p7 protein of HCV displays membrane channel activity in vitro and is essential for replication in vivo though its precise role in the virus life cycle is unknown. p7 channel activity can be specifically inhibited by several classes of compounds, making this protein an attractive candidate for drug development, though techniques used to date in characterising this protein are unsuited to compound library screening. Here we describe an assay for the channel forming ability of p7 based on the release of a fluorescent indicator from liposomes. We show that recombinant p7 from genotype 1b HCV causes a dose-dependent release of dye when mixed with liposomes and that this property is enhanced at acidic pH. We demonstrate that this activity is due to the formation of a size-selective pore rather than non-specific disruption of liposomes and that activity can be blocked by amantadine and several other compounds, validating it as a measure of p7 channel function. This system provides the first convenient in vitro assay for exploiting p7 as a therapeutic target.

Evidence for the formation of a heptameric ion channel complex by the hepatitis C virus p7 protein in vitro.

The p7 protein of hepatitis C virus functions as an ion channel both in vitro and in cell-based assays and is inhibited by amantadine, long alkyl chain imino-sugar derivatives, and amiloride compounds. Future drug design will be greatly aided by information on the stoichiometry and high resolution structure of p7 ion channel complexes. Here, we have refined a bacterial expression system for p7 based on a glutathione S-transferase fusion methodology that circumvents the inherent problems of hydrophobic protein purification and the limitations of chemical synthesis. Rotational averaging and harmonic analysis of transmission electron micrographs of glutathione S-transferase-FLAG-p7 fusion proteins in liposomes revealed a heptameric stoichiometry. The oligomerization of p7 protein was then confirmed by SDS-PAGE and mass spectrometry analysis of pure, concentrated FLAG-p7. The same protein was also confirmed to function as an ion channel in suspended lipid bilayers and was inhibited by amantadine. These data validate this system as a means of generating high resolution structural information on the p7 ion channel complex.

Signal peptide cleavage and internal targeting signals direct the hepatitis C virus p7 protein to distinct intracellular membranes.

The hepatitis C virus (HCV) p7 protein forms an amantadine-sensitive ion channel required for viral replication in chimpanzees, though its precise role in the life cycle of HCV is unknown. In an attempt to gain some insights into p7 function, we examined the intracellular localization of p7 using epitope tags and an anti-p7 peptide antibody, antibody 1055. Immunofluorescence labeling of p7 at its C terminus revealed an endoplasmic reticulum (ER) localization independent of the presence of its signal peptide, whereas labeling the N terminus gave a mitochondrial-type distribution in brightly labeled cells. Both of these patterns could be visualized within individual cells, suggestive of separate pools of p7 where the N and C termini differed in accessibility to antibody. These patterns were disrupted by preventing signal peptide cleavage. Subcellular fractionation revealed that p7 was enriched in a heavy membrane fraction associated with mitochondria as well as normal ER-derived microsomes. The complex regulation of the intracellular distribution of p7 suggests that p7 plays multiple roles in the HCV life cycle either intracellularly or as a virion component.

A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria.

We previously identified the function of the hepatitis C virus (HCV) p7 protein as an ion channel in artificial lipid bilayers and demonstrated that this in vitro activity is inhibited by amantadine. Here we show that the ion channel activity of HCV p7 expressed in mammalian cells can substitute for that of influenza virus M2 in a cell-based assay. This was also the case for the p7 from the related virus, bovine viral diarrhoea virus (BVDV). Moreover, amantadine was shown to abrogate HCV p7 function in this assay at a concentration that specifically inhibits M2. Mutation of a conserved basic loop located between the two predicted trans-membrane alpha helices rendered HCV p7 non-functional as an ion channel. The intracellular localization of p7 was unaffected by this mutation and was found to overlap significantly with membranes associated with mitochondria. Demonstration of p7 ion channel activity in cellular membranes and its inhibition by amantadine affirm the protein as a target for future anti-viral chemotherapy.

The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine.

Hepatitis C virus (HCV) cannot be grown in vitro, making biochemical identification of new drug targets especially important. HCV p7 is a small hydrophobic protein of unknown function, yet necessary for particle infectivity in related viruses [Harada, T. et al., (2000) J. Virol. 74, 9498-9506]. We show that p7 can be cross-linked in vivo as hexamers. Escherichia coli expressed p7 fusion proteins also form hexamers in vitro. These and HIS-tagged p7 function as calcium ion channels in black lipid membranes. This activity is abrogated by Amantadine, a compound that inhibits ion channels of influenza [Hay, A.J. et al. (1985) EMBO J. 4, 3021-3024; Duff, K.C. and Ashley, R.H. (1992) Virology 190, 485-489] and has recently been shown to be active in combination with current HCV therapies.