The humoral immune response to SARS‐CoV‐2 results in antibodies against spike (S) and nucleoprotein (N). However, whilst there are widely available neutralization assays for S antibodies, there is no assay for N‐antibody activity. Here, we present a simple in vitro method called EDNA (electroporated‐antibody‐dependent neutralization assay) that provides a quantitative measure of N‐antibody activity in unpurified serum from SARS‐CoV‐2 convalescents. We show that N antibodies neutralize SARS‐CoV‐2 intracellularly and cell‐autonomously but require the cytosolic Fc receptor TRIM21. Using EDNA, we show that low N‐antibody titres can be neutralizing, whilst some convalescents possess serum with high titres but weak activity. N‐antibody and N‐specific T‐cell activity correlates within individuals, suggesting N antibodies may protect against SARS‐CoV‐2 by promoting antigen presentation. This work highlights the potential benefits of N‐based vaccines and provides an in vitro assay to allow the antibodies they induce to be tested.

The intracellular recycling of ascorbic acid from dehydroascorbic acid by the glutathione-glutathione reductase system has been well-characterized. We propose that extracellular recycling of ascorbic acid is performed in a similar manner by cysteine-rich, glutathione-like regions of the first and second extracellular loops of some aminergic receptors including adrenergic, histaminergic, and dopaminergic receptors. Previous research in our laboratory demonstrated that ascorbic acid binds to these receptors at a site on their first or second extracellular loops, significantly enhancing ligand activity, and apparently recycling hundreds of times their own concentration of ascorbate in an enzymatic fashion. In this study, we have synthesized 25 peptides from the first and second extracellular loops of aminergic and insulin receptors and compared them directly to glutathione for their ability to prevent the oxidation of ascorbate and to regenerate ascorbate from dehydroascorbic acid. Peptide sequences that mimic glutathione in containing a cysteine and a glutamic acid-like amino acid also mimic glutathione activity in effects and in kinetics. Some (but not all) peptide sequences that contain one or more methionines instead of cysteine can significantly retard the oxidation of ascorbic acid but do not recycle it from dehydroascorbate into ascorbate. Peptides lacking both cysteines and methionines uniformly failed to alter significantly ascorbate or dehydroascorbate oxidation or reduction. We believe that this is the first proof that receptors may carry out both ligand binding and enzymatic activity extracellularly. Our results suggest the existence of a previously unknown extracellular system for recycling ascorbate. Copyright © 2016 John Wiley & Sons, Ltd.

Diet composition, calories, and fasting times contribute to the maintenance of health. However, the impact of very low-calorie intake (VLCI) achieved with either standard laboratory chow (SD) or a plant-based fasting mimicking diet (FMD) is not fully understood. Here, using middle-aged male mice we show that 5 months of short 4:10 VLCI cycles lead to decreases in both fat and lean mass, accompanied by improved physical performance and glucoregulation, and greater metabolic flexibility independent of diet composition. A long-lasting metabolomic reprograming in serum and liver is observed in mice on VLCI cycles with SD, but not FMD. Further, when challenged with an obesogenic diet, cycles of VLCI do not prevent diet-induced obesity nor do they elicit a long-lasting metabolic memory, despite achieving modest metabolic flexibility. Our results highlight the importance of diet composition in mediating the metabolic benefits of short cycles of VLCI.

The causes of insulin resistance are not well-understood in either type 1 or type 2 diabetes. Insulin (INS) is known to undergo rapid non-enzymatic covalent conjugation to glucose or other sugars (glycation). Because the insulin receptor (IR) has INS-like regions associated with both glucose and INS binding, we hypothesize that hyperglycemic conditions may rapidly glycate the IR, chronically interfering with INS binding. IR peptides were synthesized spanning IR- associated INS-binding regions. Glycation rates of peptides under hyperglycemic conditions were followed over six days using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. INS conjugated to horse-radish peroxidase was used to determine INS binding to IR peptides in glycated and non-glycated forms. Several IR peptides were glycated up to 14% within days of exposure to 20–60 mM glucose. Rates of IR-peptide glycation were comparable to those of insulin. Glycation of four IR peptides significantly inhibits INS binding to them. Glycation of intact IR also decreases INS binding by about a third, although it was not possible to confirm the glycation sites on the intact IR. Glycation of the IR may therefore provide a mechanism by which INS resistance develops in diabetes. Demonstration of glycation of intact IR in vivo is needed.