Redox stress induces DNA mutations that contribute to chronic conditions affecting human health and to the emergence of antibiotic resistance. Yet, the impact of redox stress-induced mutagenesis remains difficult to decipher because redox agents are diverse and produce hard-to-detect mutational outcomes. Single-stranded DNA (ssDNA) provides a useful tool for studying mutagenic effects of redox agents, as it is particularly susceptible to damage and cannot be repaired by most DNA repair pathways. Here, we established a protocol to investigate redox stress-induced mutagenesis based on the Escherichia coli conjugative ssDNA that is transferred from donor to recipient cells. Using the environmentally relevant redox agents, potassium bromate and hydrogen peroxide, we show that the F episome is remarkably sensitive to weak mutagens during conjugation, enabling the detection of significant differences in mutational spectra induced by these agents. We support our findings with metabolomic analysis, which reveals agent-specific responses in E. coli . We compare these results with those obtained using a yeast ssDNA reporter and conclude that redox-induced mutagenesis depends, among other factors, on the metabolic context of the analysed system. These findings have important implications because the high sensitivity of conjugation-associated ssDNA to environmental mutagens may contribute to the evolution of antibiotic resistance. GRAPHICAL ABSTRACT:

BACKGROUND:Most peanut allergic patients have antibodies to Ara h 1. Many studies have focused on the C-terminal vicilin region, with less attention concentrated on the N-terminal α-hairpinin domain. This study evaluated human monoclonal antibodies targeted to both regions. METHODS:Microarrays of Ara h 1 peptides were probed for serum IgE binding. Human monoclonal antibodies to Ara h 1 were discovered from single-cell sequencing and hybridoma technologies. Western blots evaluated the cleavage pattern of Ara h 1 in peanuts. A mouse model of passive systemic anaphylaxis (PSA) evaluated the functional coupling of antibodies. ELISAs established antibody specificities. RESULTS:In 75 allergic patients, the most frequently recognized peptides from Ara h 1 were in the unstructured region (91%), the α-hairpinin (86%), and the vicilin region (79%). Monoclonal antibodies were divided by specificity for the α-hairpinin and vicilin regions with no significant cross-reactivity observed. The anti-α-hairpinin mAb 40C7 blocked 55%-92% of patient IgE to the domain in seven patients; ELISA results showed the epitope of 40C7 was associated with a frequently recognized peptide found via microarray. Pairing of anti-α-hairpinin mAbs failed to induce anaphylaxis via PSA; however, a single mAb against the vicilin region induced anaphylaxis when challenged with nAra h 1. CONCLUSIONS:The α-hairpinin of Ara h 1 contains a dominant epitope that is both frequently recognized and blocks a majority of the IgE against this domain. Including this region in tests may improve diagnosis of peanut allergy. The trimeric structure of the vicilin region explains the ability of Ara h 1 to induce anaphylaxis with a single antibody.

BACKGROUND AND OBJECTIVES:Nonspecific lipid transfer proteins (nsLTPs) are frequently cross-reactive allergens that hamper diagnosis and avoidance. It is challenging to distinguish cross-reactivity from cosensitization with polyclonal serum owing to the presence of a few promiscuous antibodies or many highly specific antibodies. Objective: We hypothesized that a robust analysis of more human monoclonal antibodies (mAbs) would enable us to compare crossreactivity with cosensitization. METHODS:Human monoclonal antibodies were cloned from allergic patients via single cell sequencing and screened for affinity to extracts and recombinant allergens. Ara h 9 was expressed and crystallized with the mAb IGX-3103. Affinity for nsLTPs was explored using molecular modeling, site-directed mutagenesis, and ELISA. RESULTS:A human IgG4 mAb named IGX-3103 was discovered from a type 2-polarized memory B cell expressing CD23, IL-4Ra, and germline IGHE. IGX-3103 bound to 19 different type 1 nsLTP allergens and to extracts from sources without a characterized nsLTP allergen. The structure showed that IGX-3103 induced a conformational change in Ara h 9, enabling a hydrophobic residue from the antibody, Phe104, to enter the lipid binding cavity. Key residues in the epitope were identified to include Leu1, Ser2, Cys3, Lys39, and Asp43 in Ara h 9; these residues are conserved across type 1 nsLTPs, thus explaining the promiscuity of IGX-3103. CONCLUSIONS:IGX-3103 is an example of a human mAb with cross-reactivity to pollen, fruit, and seed type 1 nsLTPs. This observation anecdotally supports the possibility that a few promiscuous mAbs could be driving cross-reactivity.

Introduction:Individuals allergic to peanuts (PN) may show IgE cross-reactivity to tree nuts, especially walnuts (WN), which often complicates diagnosis. Vicilin-buried peptides (VBPs), short segments within the N-terminal vicilin leader sequence (LS), contribute to cross-reactivity due to their ubiquitous, highly conserved and stable α-hairpin structures. The binding patterns of cross-reactive IgE to linear and conformational epitopes of PN and WN LSs and constituent VBPs may serve as a model for understanding clinically symptomatic cross-reactivity. Methods:Serum samples (n = 30) from primarily oral food challenge-positive individuals with PN allergy (PNA, 33%), WN allergy (WNA, 47%), and PN and WN allergies (PWA, 20%) were collected. These sera and a monoclonal IgE antibody (6D12) were examined for IgE binding with microarrays of overlapping peptides from native Ara h 1 LS [AH1LS, Ara h 1.0101 (26-84)] and recombinant Jug r 2 LS [JR2LS, Jug r 2.0101 (1-173)] and via direct and competitive inhibition ELISA with intact LSs and constituent VBPs from PN (AH1.1) and WN (JR2.1, JR2.2, JR2.3). A mixed model analysis assessed the contribution of IgE binding patterns to VBPs in relation to PNA, WNA, or PWA status. Results:All three intact WN VBPs bound IgE at similar frequencies, with individual sera showing varying preferences for specific VBPs. AH1.1 was less recognized by WNA individuals but more frequently recognized by PNA and PWA subjects. WN VBPs were recognized by PNA sera samples at rates comparable to AH1.1. Our data indicates that each VBP can bind to one IgE molecule with high affinity. In a competitive inhibition ELISA, combining VBP competitors did not enhance inhibition compared to the dominant VBP, suggesting that both high- and low-affinity VBPs compete for the same monoclonal IgE in serum. This observation was mimicked by 6D12, a monoclonal IgE against JR2.1. Discussion:Cross-reactivity among VBPs most likely arises from monoclonal IgE binding to α-hairpin structures and their overlapping linear amino acid sequences. The combination of linear and conformational IgE binding patterns enabled us to differentiate between the WNA, PNA, and PWA groups in this study and may assist us in using AH1LS and JR2LS to distinguish PN and WN allergies in the future.

Molecular analysis of interactions between IgE antibody and allergen allows the structural basis of IgE recognition to be defined. Human IgE (hIgE) epitopes of respiratory lipocalin allergens, including Can f 1, remain elusive due to a lack of IgE-allergen complexes. This study aims to map the structure of allergenic epitopes on Can f 1. The fragment antigen-binding (Fab) regions of Can f 1 specific human IgE monoclonal antibodies (hIgE mAb) were used to determine the structures of IgE epitopes. Epitope mutants were designed to target Can f 1 epitopes. Immunoassays and a human FcεRIα transgenic mouse model of passive anaphylaxis in vivo were used to assess the functional activity of epitope mutants. Crystal structures of natural or recombinant Can f 1 complexed with two hIgE mAb 1J11 and 12F3 Fabs, respectively, were determined. The hIgE mAb bound to two partially overlapping epitopes and recognized two different Can f 1 conformations. The hIgE mAb 12F3 showed an unusual mode of binding by protruding its heavy chain CDR3 inside the Can f 1 calyx. Epitope mutants generated based on the structural analyses displayed a 64%-89% reduction in IgE antibody binding and failed to induce passive anaphylaxis in a human FcεRIα transgenic mouse model. In summary, the structures of Can f 1-hIgE Fab complexes revealed two unique and partially overlapping epitopes on Can f 1. The modification of the identified IgE epitopes provides a pathway for the design of hypoallergens to treat dog allergies.

Metformin (Met) and liraglutide (Lira) are preferred diabetes therapies that may improve glycemia by modulating the gut microbiome, but the mechanisms and pathways are unknown and few data exist in youth-onset type 2 diabetes (Y-T2D). In a 3-month parallel clinical trial in African American Y-T2D randomized to Met (n = 14) or Met+Lira (n = 11), we compared gut microbial composition and metabolomic profiles and determined the relationship of changes in microbial abundance with glycemia and plasma metabolites. After 3 months, Met was associated with greater relative abundance of Eubacterium and Eubacterium rectale and lower Bacteroides ovates (p < 0.05). Met+Lira was associated with greater Bacteroides fragilis and lower Streptococcus thermophilus (p < 0.05). Met group had increased (>1.5-fold) plasma cholic secondary bile acids (sulfochenodeoxycholic acid, nutriacholic acid, alpha-muricholic acid, and C24 dihydroxy bile acid; p ≤ 0.002). The change in nutriacholic acid correlated with lower fasting glucose (r = -0.7, p < 0.05). Shifts in microbiota taxa were not associated with plasma short-chain fatty acids (SCFA), hemoglobin A1c or glucose. Short-term Met and Met+Lira in Y-T2D were related to distinct shifts toward bile acid and SCFA-producing gut microbiota taxa, and secondary bile acid metabolites correlated with improved glycemia, suggesting bile acid pathways may be important modulators of glycemia in youth on metformin.Clinical trials.gov identifier: NCT02960659.

Sustained clinical tolerance to food allergens can be induced by oral immunotherapy (OIT). We previously identified neutralizing IgG4 antibodies (nAbs) as key players in disrupting allergen-IgE binding . Furthermore, using an inhibitory ELISA, we used a combination of two nAbs to identify that the induction of nAbs occurs during OIT only in those with sustained tolerance. Here, we seek to develop a quantitative assay to test the utility of nAbs as an early biomarker of allergic tolerance. We will compare nAbs using protein G purified plasma from OIT-treated peanut allergic patients with sustained and non-sustained tolerance. Using previously identified mAbs, we developed a flow cytometry bead-based assay (BBNA) using streptavidin coated beads and biotinylated mutated recombinant Arah2 (rArah2). Quantitative fluorescent flow cytometry beads were used for antibody quantitation. We validated the use of mrArah2 using biolayer interferometry and mAbs. The BBNA is highly sensitive and reproducible (CV-2.5%). The BBNA correlates with previously published ELISA (R2=0.9) data. The induction of IgG4 nAbs occurs only in sustained tolerance by BBNA (P < 0.01). A quantitative, sample-sparing BBNA assay assessing direct antibody binding to rArah2 reliably identifies serum nAbs in OIT-treated allergic patients. OIT-induced nAbs occurs only in sustained responses. Therefore, nAbs BBNA could be a valuable serum biomarker to predict long term clinical tolerance in peanut oral immunotherapy. NIAID, NIH (5R01AI155630, 1R21AI159732, to SUP); NIEHS, NIH (1ZIAES102906, to GAM; 1ZICES102645, to LCP); Food Allergy Science Initiative award (SUP). The clinical trial work at Harvard Clinical and Translational Science Center (1UL1TR001102 and 8UL1TR000170) from the National Center for Advancing Translational Science, and 1UL1 RR025758 from the National Center for Research Resources. Immune Mechanisms of Human Disease (HUM)

Humans develop immunoglobulin G (IgG) antibodies to the foods they consume. In the context of food allergy, allergen-specific IgG antibodies can sequentially class-switch to pathogenic IgE. However, the mechanism underlying the antigenicity of food proteins remains uncharacterized. Here, we identified convergent antibodies arising from different antibody gene rearrangements that bind to the immunodominant peanut allergen Ara h 2 and characterized allelic and junctional constraints on germline antibody specificity. Structurally, we found similar epitope-paratope interactions across multiple gene rearrangements. We demonstrate that these germline-encoded epitope-specific convergent antibodies to peanut occur commonly in the population because of the worldwide prevalence of the relevant gene rearrangements, allelic independence, and junctional malleability. As a result, serum IgG to this public epitope is prevalent among diverse cohorts of nonallergic peanut-consuming infants and peanut-allergic children and adults. This work demonstrates that IgG recognition of dietary antigens can be intrinsically programmed by the germline antibody repertoire.

Allergen-specific immunotherapy represents the only method of achieving a lasting reduction in the severity of allergic symptoms. However, the need to expose patients to the allergens to which they are sensitized carries risks. One solution is to use denatured allergens whereby the structure of allergenic proteins is disrupted, preventing their recognition by immunoglobulin E (IgE) antibodies and thus reducing the risk of adverse reactions. Denaturation is often carried out by using chemical cross-linking to generate allergoids. Gold nanoclusters (AuNCs) are emerging as versatile tools in biotechnology due in part to their ability to conjugate a wide range of biological molecules. Previous works have described the formation of AuNC using egg allergens such as Gal d 4 (lysozyme), Gal d 2 (ovalbumin), and whole egg whites. In all cases, AuNC bioconjugation disrupted the protein structure, allowing for their use in biosensing applications. In this work, we hypothesize that these AuNC-allergen bioconjugates could be used to generate "Allergolds", chemically altered versions of allergenic proteins analogous to traditional allergoid formulations. Using spectroscopic techniques, we confirm that the formation of AuNC bioconjugates of the chicken egg Gal d 4 and Gal d 2 disrupts protein structure when generated from both purified protein and whole egg whites. This structural perturbation was found to be resilient to a range of chemical conditions and successfully disrupted recognition by human IgE. These results establish Allergolds as a potential tool for generating systematically denatured allergens from both purified proteins and biological extracts.

Coronaviruses evade detection by the host immune system with the help of the endoribonuclease Nsp15, which regulates levels of viral double stranded RNA by cleaving 3 ' of uridine (U). While prior structural data shows that to cleave double stranded RNA, Nsp15's target U must be flipped out of the helix, it is not yet understood whether Nsp15 initiates flipping or captures spontaneously flipped bases. We address this gap by designing fluorinated double stranded RNA substrates that allow us to directly relate a U's sequence context to both its tendency to spontaneously flip and its susceptibility to cleavage by Nsp15. Through a combination of nuclease assays, 19F NMR spectroscopy, mass spectrometry, and single particle cryo-EM, we determine that Nsp15 acts most efficiently on unpaired Us, particularly those that are already flipped. Across sequence contexts, we find Nsp15's cleavage efficiency to be directly related to that U's tendency to spontaneously flip. Overall, our findings unify previous characterizations of Nsp15's cleavage preferences, and suggest that activity of Nsp15 during infection is partially driven by bulged or otherwise relatively accessible Us that appear at strategic positions in the viral RNA.

Hypertension is estimated to affect almost 1 billion people globally and significantly increases risk of myocardial infarction, heart failure, stroke, retinopathy and kidney disease. One major front line therapy that has been used for over 50 years involves L-type Ca 2+ channel blockers (LCCBs). One class of LCCBs is the dihydropyridine family, with amlodipine being widely prescribed regardless of gender, race, ethnicity or age. In 2020, Johnson et al. 7 reported that all LCCBs significantly increased the risk of heart failure, and attributed this effect to non-canonical activation of store-operated Ca 2+ entry. A major approach on which they based many of their arguments was to measure cytosolic Ca 2+ using the fluorescent Ca 2+ indicator dye fura-2. We recently demonstrated that amlodipine is highly fluorescent within cells and overwhelms the fura-2 signal, precluding the use of the indicator dye with amlodipine 24 . Our meta-analyses and prospective real world study showed that dihydropyridines were not associated with an increase in heart failure, likely explained by the lack of consideration by Johnson et al. 7 of well-known confounding factors such as age, race, obesity, prior anti-hypertensive treatment or diabetes 24 . Trebak and colleagues have responded to our paper with a forthright and unwavering defence of their work 27 . In this paper, we carry out a forensic dissection of Johnson et al., 7 and conduct new experiments that address directly points raised by Trebak et al. 27 . We show that there are major flaws in the design and interpretation of their key experiments, that fura-2 cannot be used with amlodipine, that there are fundamental mathematical misunderstandings and mistakes throughout their study leading to critical calculations on heart failure that are demonstrably wrong, and several of their own results are inconsistent with their interpretation. We therefore believe the study by Johnson et al. 7 is flawed at many levels and we stand by our conclusions.

INTRODUCTION:Adverse reactions are relatively common during peanut oral immunotherapy. To reduce the risk to the patient, some researchers have proposed modifying the allergen to reduce IgE reactivity, creating a putative hypoallergen. Analysis of recently cloned human IgG from patients treated with peanut immunotherapy suggested that there are three common conformational epitopes for the major peanut allergen Ara h 2. We sought to test if structural information on these epitopes could indicate mutagenesis targets for designing a hypoallergen and evaluated the reduction in IgE binding via immunochemistry and a mouse model of passive cutaneous anaphylaxis (PCA).METHODS:X-ray crystallography characterized the conformational epitopes in detail, followed by mutational analysis of key residues to modify monoclonal antibody (mAb) and serum IgE binding, assessed by ELISA and biolayer interferometry. A designed Ara h 2 hypoallergen was tested for reduced vascularization in mouse PCA experiments using pooled peanut allergic patient serum.RESULTS:A ternary crystal structure of Ara h 2 in complex with patient antibodies 13T1 and 13T5 was determined. Site-specific mutants were designed that reduced 13T1, 13T5, and 22S1 mAbs binding by orders of magnitude. By combining designed mutations from the three major conformational bins, a hexamutant (Ara h 2 E46R, E89R, E97R, E114R, Q146A, R147E) was created that reduced IgE binding in serum from allergic patients. Further, in the PCA model where mice were primed with peanut allergic patient serum, reactivity upon allergen challenge was significantly decreased using the hexamutant.CONCLUSION:These studies demonstrate that prior knowledge of common conformational epitopes can be used to engineer reduced IgE reactivity, an important first step in hypoallergen design.