Novel post-translationally cleaved Ara h 2 proteoforms: Purification, characterization and IgE-binding properties.

The 2S albumins Ara h 2 and Ara h 6 have been shown to be the most important source of allergenicity in peanut. Several isoforms of these allergens have been described. Using extraction and liquid chromatography we isolated proteins with homology to Ara h 2 and characterized hitherto unknown Ara h 2 proteoforms with additional post-translational cleavage. High-resolution mass spectrometry located the cleavage site on the non-structured loop of Ara h 2 while far UV CD spectroscopy showed a comparable structure to Ara h 2. The cleaved forms of Ara h 2 were present in genotypes of peanut commonly consumed. Importantly, we revealed that newly identified Ara h 2 cleaved proteoforms showed comparable IgE-binding using sera from 28 peanut-sensitized individuals, possessed almost the same IgE binding potency and are likely similarly allergenic as intact Ara h 2. This makes these newly identified forms relevant proteoforms of peanut allergen Ara h 2.

The Fate of IgE Epitopes and Coeliac Toxic Motifs during Simulated Gastrointestinal Digestion of Pizza Base.

Understanding how food processing may modify allergen bioaccessibility and the evolution of immunologically active peptides in the gastrointestinal tract is essential if knowledge-based approaches to reducing the allergenicity of food are to be realised. A soy-enriched wheat-based pizza base was subjected to in vitro oral-gastro-duodenal digestion and resulting digests analysed using a combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The digestion profile of pizza base resembled that of bread crust where higher temperatures during baking reduced protein solubility but still resulted in the generation of a complex mixture of peptides. MS profiling showed numerous peptides carrying IgE epitopes, and coeliac toxic motifs were in excess of 20-30 residues long and were only released after either 120 min of gastric digestion or a combination of gastric and duodenal digestion. In silico prediction tools showed an overestimated number of cleavage sites identified experimentally, with low levels of atypical peptic and chymotryptic cleavage sites identified particularly at glutamine residues. These data suggest that such alternative pepsin cleavage sites may play a role in digestion of glutamine-rich cereal foods. They also contribute to efforts to provide benchmarks for mapping in vitro digestion products of novel proteins which form part of the allergenicity risk assessment.

Purification and Initial Characterization of Ara h 7, a Peanut Allergen from the 2S Albumin Protein Family.

2S albumins are important peanut allergens. Within this protein family, Ara h 2 and Ara h 6 have been described in detail, but Ara h 7 has received little attention. We now describe the first purification of Ara h 7 and its characterization. Two Ara h 7 isoforms were purified from peanuts. Mass spectrometry revealed that both the isoforms have a post-translation cleavage, a hydroxyproline modification near the N-terminus, and four disulfide bonds. The secondary structure of both Ara h 7 isoforms is highly comparable to those of Ara h 2 and Ara h 6. Both Ara h 7 isoforms bind IgE, and Ara h 7 is capable of inhibiting the binding between Ara h 2 and IgE, suggesting at least partially cross-reactive IgE epitopes. Ara h 7 was found in all main market types of peanut, at comparable levels. This suggests that Ara h 7 is a relevant allergen from the peanut 2S albumin protein family.

Functional outcomes at 12 months for patients with traumatic brain injury, intracerebral haemorrhage and subarachnoid haemorrhage treated in an Australian neurocritical care unit: A prospective cohort study.

BACKGROUND: Acute severe brain injury is associated with significant morbidity and mortality. Patients and their families need accurate information regarding expected outcomes. Few studies have reported the long-term functional outcome of patients with acute severe brain injury treated in an Australian neurocritical care unit. OBJECTIVE: The objective of this study was to describe 12-month functional outcomes (using the extended Glasgow Outcome Scale) of patients with acute severe brain injury treated in an Australian neurocritical care unit. METHODS: This was a single-centre prospective cohort study. Patients with a diagnosis of traumatic brain injury, subarachnoid haemorrhage or intracranial haemorrhage admitted between 2015 and 2019 were enrolled. RESULTS: In total, 915 participants were enrolled during the 51-month study period. Of the cohort, 403 (44%) were admitted after traumatic brain injury, 274 (30%) after subarachnoid haemorrhage and 238 (26%) after intracranial haemorrhage. The median duration of intensive care admission was 5 days (interquartile range: 2-13), 458 (50%) received invasive ventilation, 417 (46%) received vasopressor support and 286 (31%) received an external ventricular drain. At discharge from intensive care, 150 of 915 (16.4%) had died, and the in-hospital mortality was seen in 191 of 915 patients (20.9%). Favourable functional outcome, as defined by an extended Glasgow Outcome Scale score of 5-8, was reported in 358 of available 795 patients (45.0%) at six months and in 311 of 672 available patients (46.3%) at 12 months. Those with intracranial haemorrhage reported the highest rates of unfavourable outcomes with 112 of 166 patients (67.4%) at 12 months. CONCLUSIONS: In this selected population, admission to a neurocritical care unit was associated with significant resource use. At 12 months after admission, almost half of those admitted to an Australian neurocritical unit with traumatic brain injury, subarachnoid haemorrhage and intracerebral haemorrhage report a good functional outcome.

A comparison of manual pupil examination versus an automated pupillometer in a specialised neurosciences intensive care unit.

BACKGROUND: The assessment of pupil size and reaction to light is a fundamental part of the neurological assessment; however, manual examination is prone to inaccuracies. The use of an automated infrared pupillometer is one strategy to limit error in pupil examination. OBJECTIVE: The main objective was to assess agreement between manual examination and examination using an automated infrared pupillometer in relation to pupil reaction and size in a specialised neurosciences intensive care unit. METHODS: We conducted a single-centre prospective observational study in a specialised tertiary neurosciences intensive care unit. Participants' pupils were examined hourly for 24 h by both manual examination using a pen torch and examination using an automated infrared pupillometer. RESULTS: Twenty-two participants were enrolled. A total of 935 paired pupil observations were obtained for both pupil reaction and size. There was no statistically significant disagreement in assessing pupil reaction (McNemar's test p = 0.106). Percentage agreement was 96.68% for pupil reaction, with Kappa coefficient, 0.841 (95% confidence interval: 0.7864-0.8956). For all participants, the mean difference in pupil size was 0.154 mm, with limits of agreement of -1.294 mm to +1.603 mm. CONCLUSION: There was no significant disagreement between manual and automated pupillometer observations for pupil reaction. The mean difference in measurement of pupil size was small.

Purification and Characterization of Naturally Occurring Post-Translationally Cleaved Ara h 6, an Allergen That Contributes Substantially to the Allergenic Potency of Peanut.

The 2S albumin Ara h 6 is one of the most important peanut allergens. A post-translationally cleaved Ara h 6 (pAra h 6) was purified from Virginia type peanuts, and the cleavage site was mapped using high-resolution mass spectrometry. Compared to intact Ara h 6, pAra h 6 lacks a 5-amino acid stretch, resembling amino acids 43-47 (UniProt accession number Q647G9) in the nonstructured loop. Consequently, pAra h 6 consists of two chains: an N-terminal chain of approximately 5 kDa and a C-terminal chain of approximately 9 kDa, held together by disulfide bonds. Intermediate post-translationally cleaved products, in which this stretch is cleaved yet still attached to one of the subunits, are also present. The secondary structure and immunoglobulin E (IgE) binding of pAra h 6 resembles that of intact Ara h 6, indicating that the loss of the nonstructured loop is not critical for maintaining the protein structure. Commercially available monoclonal and polyclonal immunoglobulin G (IgG) antibodies directed to Ara h 6 react with both intact Ara h 6 and pAra h 6, suggesting that the involved epitopes are not located in the area that is post-translationally cleaved. No differences between intact Ara h 6 and pAra h 6 in terms of IgE binding were found, suggesting that the area that is post-translationally cleaved is not involved in IgE epitopes either. For all main cultivars Runner, Virginia, Valencia, and Spanish, intact Ara h 6 and pAra h 6 occur in peanut at similar levels, indicating that pAra h 6 is a consistent and important contributor to the allergenic potency of peanut.

Legal Origin and Social Solidarity: The Continued Relevance of Durkheim to Comparative Institutional Analysis.

By using the classic works of Durkheim as a theoretical platform, this research explores the relationship between legal systems and social solidarity. We found that certain types of civil law system, most notably those of Scandinavia, are associated with higher levels of social capital and better welfare state provision. However, we found the relationship between legal system and societal outcomes is considerably more complex than suggested by currently fashionable economistic legal origin approaches, and more in line with the later writings of Durkheim, and, indeed, the literature on comparative capitalisms. Relative communitarianism was strongly affected by relative development, reflecting the complex relationship between institutions, state capabilities and informal social ties and networks.

Patterns of IgE responses to multiple allergen components and clinical symptoms at age 11 years.

BACKGROUND: The relationship between sensitization to allergens and disease is complex. OBJECTIVE: We sought to identify patterns of response to a broad range of allergen components and investigate associations with asthma, eczema, and hay fever. METHODS: Serum specific IgE levels to 112 allergen components were measured by using a multiplex array (Immuno Solid-phase Allergen Chip) in a population-based birth cohort. Latent variable modeling was used to identify underlying patterns of component-specific IgE responses; these patterns were then related to asthma, eczema, and hay fever. RESULTS: Two hundred twenty-one of 461 children had IgE to 1 or more components. Seventy-one of the 112 components were recognized by 3 or more children. By using latent variable modeling, 61 allergen components clustered into 3 component groups (CG1, CG2, and CG3); protein families within each CG were exclusive to that group. CG1 comprised 27 components from 8 plant protein families. CG2 comprised 7 components of mite allergens from 3 protein families. CG3 included 27 components of plant, animal, and fungal origin from 12 protein families. Each CG included components from different biological sources with structural homology and also nonhomologous proteins arising from the same biological source. Sensitization to CG3 was most strongly associated with asthma (odds ratio [OR], 8.20; 95% CI, 3.49-19.24; P < .001) and lower FEV1 (P < .001). Sensitization to CG1 was associated with hay fever (OR, 12.79; 95% CI, 6.84-23.90; P < .001). Sensitization to CG2 was associated with both asthma (OR, 3.60; 95% CI, 2.05-6.29) and hay fever (OR, 2.52; 95% CI, 1.38-4.61). CONCLUSIONS: Latent variable modeling with a large number of allergen components identified 3 patterns of IgE responses, each including different protein families. In 11-year-old children the pattern of response to components of multiple allergens appeared to be associated with current asthma and hay fever but not eczema.

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.

Rapid fingerprinting of milk thermal processing history by intact protein mass spectrometry with nondenaturing chromatography.

Thermal processing of foods results in proteins undergoing conformational changes, aggregation, and chemical modification notably with sugars via the Maillard reaction. This can impact their functional, nutritional, and allergenic properties. Native size-exclusion chromatography with online electrospray mass spectrometry (SEC-ESI-MS) was used to characterize processing-induced changes in milk proteins in a range of milk products. Milk products could be readily grouped into either pasteurized liquid milks, heavily processed milks, or milk powders by SEC behavior, particularly by aggregation of whey proteins by thermal processing. Maillard modification of all major milk proteins by lactose was observed by MS and was primarily present in milk powders. The method developed is a rapid tool for fingerprinting the processing history of milk and has potential as a quality control method for food ingredient manufacture. The method described here can profile milk protein oligomeric state, aggregation, and Maillard modification in a single shot, rapid analysis.

Current perspectives and recommendations for the development of mass spectrometry methods for the determination of allergens in foods.

Allergen detection and quantification is an essential part of allergen management as practiced by food manufacturers. Recently, protein MS methods (in particular, multiple reaction monitoring experiments) have begun to be adopted by the allergen detection community to provide an alternative technique to ELISA and PCR methods. MS analysis of proteins in foods provides additional challenges to the analyst, both in terms of experimental design and methodology: (1) choice of analyte, including multiplexing to simultaneously detect several biologically relevant molecules able to trigger allergic reactions; (2) choice of processing stable peptide markers for different target analytes that should be placed in publicly available databases; (3) markers allowing quantification (e.g., through standard addition or isotopically labeled peptide standards); (4) optimization of protease digestion protocols to ensure reproducible and robust method development; and (5) effective validation of methods and harmonization of results through the use of naturally incurred reference materials spanning several types of food matrix.

Effect of heating and glycation on the allergenicity of 2S albumins (Ara h 2/6) from peanut.

BACKGROUND: Peanut allergy is one of the most common and severe food allergies, and processing is known to influence the allergenicity of peanut proteins. We aimed to establish the effect of heating and glycation on the IgE-binding properties and biological activity of 2S albumins (Ara h 2/6) from peanut. METHODOLOGY/PRINCIPAL FINDINGS: Native Ara h 2/6 was purified from raw peanuts and heated in solution (15 min, 110°C) in the presence or absence of glucose. Ara h 2 and 6 were also purified from roasted peanut. Using PBMC and sera from peanut-allergic patients, the cellular proliferative potency and IgE reactivity (reverse EAST inhibition) and functionality (basophil degranulation capacity) of allergens were assessed. Heating Ara h 2/6 at 110°C resulted in extensive denaturation, hydrolysis and aggregation of the protein, whilst Ara h 2 and 6 isolated from roasted peanut retained its native conformation. Allergen stimulation of PBMC induced proliferation and Th2 cytokine secretion which was unaffected by thermal processing. Conversely, IgE reactivity and functionality of Ara h 2/6 was decreased by heating. Whilst heating-glycation further reduced the IgE binding capacity of the proteins, it moderated their loss of histamine releasing capacity. Ara h 2 and 6 purified from roasted peanut demonstrated the same IgE reactivity as unheated, native Ara h 2/6. CONCLUSIONS/SIGNIFICANCE: Although no effect of processing on T-cell reactivity was observed, heat induced denaturation reduced the IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein which may explain the allergenic potency of this protein. Through detailed molecular study and allergenicity assessment approaches, this work then gives new insights into the effect of thermal processing on structure/allergenicity of peanut proteins.

Responsiveness of the major birch allergen Bet v 1 scaffold to the gastric environment: impact on structure and allergenic activity.

SCOPE: Four Bet v 1 homologous food allergens from celeriac (rApi g 1), apple (rMal d 1), peach (rPru p 1) and hazelnut (rCor a 1), were used to probe the structural responsiveness of the Bet v 1 scaffold to gastric digestion conditions and its impact on allergenicity. METHODS AND RESULTS: Low pH induced conformational changes of all homologues, which was reduced at physiological ionic strength for all except rPru p 1 as observed by circular dichroism (CD)-spectroscopy. The homologues were rapidly digested by pepsin, losing their IgE binding activity, although the kinetics and patterns of digestion varied subtly between homologues, rApi g 1 being the most stable. We have demonstrated for the first time that gastric phosphatidyl-choline (PC) induced conformational changes in all homologues but only rMal d 1 penetrated the PC vesicles as detected by fluorescence polarization, slowing its digestion and retaining more of its allergenic activity. PC enhanced basophil activation of all digested allergens except rApi g 1. CONCLUSION: The Bet v 1 scaffold is generally susceptible to low pH and pepsinolysis and interacts with PC vesicles, properties which can explain effects of the gastric environment on their allergenicity. These data show the importance of including surfactants in model digestion systems.

Spectroscopic analysis of diversity of Arabinoxylan structures in endosperm cell walls of wheat cultivars (Triticum aestivum) in the HEALTHGRAIN diversity collection.

Fifty bread wheat (Triticum aestivum L.) cultivars were selected from the HEALTHGRAIN germplasm collection based on variation in their contents of total and water-extractable arabinoxylan. FT-IR spectroscopic mapping of thin transverse sections of grain showed variation in cell wall arabinoxylan composition between the cultivars, from consisting almost entirely of low-substituted arabinoxylan (e.g., T.aestivum 'Claire') to almost entirely of highly substituted arabinoxylan (e.g., T.aestivum 'Manital') and a mixture of the two forms (e.g., T.aestivum 'Hereward'). Complementary data were obtained using endoxylanase digestion of flour followed by HP-AEC analysis of the arabinoxylan oligosaccharides. This allowed the selection of six cultivars for more detailed analysis using FT-IR and (1)H NMR spectroscopy to determine the proportions of mono-, di-, and unsubstituted xylose residues. The results of the two analyses were consistent, showing that variation in the composition and structure of the endosperm cell wall arabinoxylan is present between bread wheat cultivars. The heterogeneity and spatial distribution of the arabinoxylan in endosperm cell walls may be exploited in wheat processing as it may allow the production of mill streams enriched in various arabinoxylan fractions which have beneficial effects on health.

High pressure, thermal and pulsed electric-field-induced structural changes in selected food allergens.

SCOPE: The effects of high-pressure/temperature treatment and pulsed electric field treatment on native peanut Ara h 2, 6 and apple Mal d 3 and Mal d 1b prepared by heterologous expression were examined. METHODS AND RESULTS: Changes in secondary structure and aggregation state of the treated proteins were characterized by circular dichroism spectroscopy and gel-filtration chromatography. Pulsed electric field treatment did not induce any significant changes in the structure of any of the allergens. High-pressure/temperature at 20 °C did not change the structure of the Ara h 2, 6 or Mal d 3 and resulted in only minor changes in structure of Mal d 1b. Ara h 2, 6 was stable to HPP at 80 °C, whereas changes in circular dichroism spectra were observed for both apple allergens. However, these changes were attributable to aggregation and adiabatic heating during HPP. An ELISA assay of temperature treated Mal d 3 showed the antibody reactivity correlated well with the loss of structure. CONCLUSION: In conclusion, novel-processing techniques had little effect on purified allergen structure. Further studies will demonstrate if these stability properties are retained in foodmatrices.

The structural characteristics of nonspecific lipid transfer proteins explain their resistance to gastroduodenal proteolysis.

The structure and stability of the allergenic nonspecific lipid transfer protein (LTP) of peach were compared with the homologous LTP1 of barley and its liganded form LTP1b. All three proteins were resistant to gastric pepsinolysis and were only slowly digested at 1 to 2 out of 14 potential tryptic and chymotryptic cleavage sites under duodenal conditions. Peach LTP was initially cleaved at Tyr79-Lys80 and then at Arg39-Thr40 (a site lost in barley LTP1). Molecular dynamics simulations of the proteins under folded conditions showed that the backbone flexibility is limited, explaining the resistance to duodenal proteolysis. Arg39 and Lys80 side chains were more flexible in simulations of peach compared with barley LTP1. This may explain differences in the rates of cleavage observed experimentally for the two proteins and suggests that the flexibility of individual amino acid side chains could be important in determining preferred proteolytic cleavage sites. In order to understand resistance to pepsinolysis, proteins were characterized by NMR spectroscopy at pH 1.8. This showed that the helical regions of both proteins remain folded at this pH. NMR hydrogen exchange studies confirmed the rigidity of the structures at acidic pH, with barley LTP1 showing some regions with greater protection. Collectively, these data suggest that the rigidity of the LTP scaffold is responsible for their resistance to proteolysis. Gastroduodenal digestion conditions do not disrupt the 3D structure of peach LTP, explaining why LTPs retain their ability to bind IgE after digestion and hence their allergenic potential.