Rationally designed immunogens enable immune focusing following SARS-CoV-2 spike imprinting

Eliciting antibodies to surface-exposed viral glycoproteins can generate protective responses that control and prevent future infections. Targeting conserved sites may reduce the likelihood of viral escape and limit the spread of related viruses with pandemic potential. Here we leverage rational immunogen design to focus humoral responses on conserved epitopes. Using glycan engineering and epitope scaffolding in boosting immunogens, we focus murine serum antibody responses to conserved receptor binding motif (RBM) and receptor binding domain (RBD) epitopes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike imprinting. Although all engineered immunogens elicit a robust SARS-CoV-2-neutralizing serum response, RBM-focusing immunogens exhibit increased potency against related sarbecoviruses, SARS-CoV, WIV1-CoV, RaTG13-CoV, and SHC014-CoV; structural characterization of representative antibodies defines a conserved epitope. RBM-focused sera confer protection against SARS-CoV-2 challenge. Thus, RBM focusing is a promising strategy to elicit breadth across emerging sarbecoviruses without compromising SARS-CoV-2 protection. These engineering strategies are adaptable to other viral glycoproteins for targeting conserved epitopes

Genetic loci inherited from hens lacking maternal behaviour both inhibit and paradoxically promote this behaviour

International audienceBackground A major step towards the success of chickens as a domesticated species was the separation between maternal care and reproduction. Artificial incubation replaced the natural maternal behaviour of incubation and, thus, in certain breeds, it became possible to breed chickens with persistent egg production and no incubation behaviour; a typical example is the White Leghorn strain. Conversely, some strains, such as the Silkie breed, are prized for their maternal behaviour and their willingness to incubate eggs. This is often colloquially known as broodiness.ResultsUsing an F2 linkage mapping approach and a cross between White Leghorn and Silkie chicken breeds, we have mapped, for the first time, genetic loci that affect maternal behaviour on chromosomes 1, 5, 8, 13, 18 and 19 and linkage group E22C19W28. Paradoxically, heterozygous and White Leghorn homozygous genotypes were associated with an increased incidence of incubation behaviour, which exceeded that of the Silkie homozygotes for most loci. In such cases, it is likely that the loci involved are associated with increased egg production. Increased egg production increases the probability of incubation behaviour occurring because egg laying must precede incubation. For the loci on chromosomes 8 and 1, alleles from the Silkie breed promote incubation behaviour and influence maternal behaviour (these explain 12 and 26 % of the phenotypic difference between the two founder breeds, respectively).ConclusionsThe over-dominant locus on chromosome 5 coincides with the strongest selective sweep reported in chickens and together with the loci on chromosomes 1 and 8, they include genes of the thyrotrophic axis. This suggests that thyroid hormones may play a critical role in the loss of incubation behaviour and the improved egg laying behaviour of the White Leghorn breed. Our findings support the view that loss of maternal incubation behaviour in the White Leghorn breed is the result of selection for fertility and egg laying persistency and against maternal incubation behaviour

Human malarial disease: a consequence of inflammatory cytokine release

Malaria causes an acute systemic human disease that bears many similarities, both clinically and mechanistically, to those caused by bacteria, rickettsia, and viruses. Over the past few decades, a literature has emerged that argues for most of the pathology seen in all of these infectious diseases being explained by activation of the inflammatory system, with the balance between the pro and anti-inflammatory cytokines being tipped towards the onset of systemic inflammation. Although not often expressed in energy terms, there is, when reduced to biochemical essentials, wide agreement that infection with falciparum malaria is often fatal because mitochondria are unable to generate enough ATP to maintain normal cellular function. Most, however, would contend that this largely occurs because sequestered parasitized red cells prevent sufficient oxygen getting to where it is needed. This review considers the evidence that an equally or more important way ATP deficency arises in malaria, as well as these other infectious diseases, is an inability of mitochondria, through the effects of inflammatory cytokines on their function, to utilise available oxygen. This activity of these cytokines, plus their capacity to control the pathways through which oxygen supply to mitochondria are restricted (particularly through directing sequestration and driving anaemia), combine to make falciparum malaria primarily an inflammatory cytokine-driven disease

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Boronic Acid with High Oxidative Stability and Utility in Biological Contexts

Despite their desirable attributes, boronic acids have had a minimal impact in biological contexts. A significant problem has been their oxidative instability. At physiological pH, phenylboronic acid and its boronate esters are oxidized by reactive oxygen species at rates comparable to those of thiols. After considering the mechanism and kinetics of the oxidation reaction, we reasoned that diminishing electron density on boron could enhance oxidative stability. We found that a boralactone, in which a carboxyl group serves as an intramolecular ligand for the boron, increases stability by 104-fold. Computational analyses revealed that the resistance to oxidation arises from diminished stabilization of the p orbital of boron that develops in the rate-limiting transition state of the oxidation reaction. Like simple boronic acids and boronate esters, a boralactone binds covalently and reversibly to 1,2-diols, such as those in saccharides. The kinetic stability of its complexes is, however, at least 20-fold greater. A boralactone also binds covalently to a serine side chain in a protein. These attributes confer unprecedented utility upon boralactones in the realms of chemical biology and medicinal chemistry.</p

Ribonuclease zymogen induces cytotoxicity upon HIV-1 infection

Abstract Background Targeting RNA is a promising yet underdeveloped modality for the selective killing of cells infected with HIV-1. The secretory ribonucleases (RNases) found in vertebrates have cytotoxic ribonucleolytic activity that is kept in check by a cytosolic ribonuclease inhibitor protein, RI. Methods We engineered amino acid substitutions that enable human RNase 1 to evade RI upon its cyclization into a zymogen that is activated by the HIV-1 protease. In effect, the zymogen has an HIV-1 protease cleavage site between the termini of the wild-type enzyme, thereby positioning a cleavable linker over the active site that blocks access to a substrate. Results The amino acid substitutions in RNase 1 diminish its affinity for RI by 106-fold and confer high toxicity for T-cell leukemia cells. Pretreating these cells with the zymogen leads to a substantial drop in their viability upon HIV-1 infection, indicating specific toxicity toward infected cells. Conclusions These data demonstrate the utility of ribonuclease zymogens as biologic prodrugs

Stereoelectronic Effects Impact Glycan Recognition

Recognition of distinct glycans is central to biology, and lectins mediate this function. Lectin glycan preferences are usually centered on specific monosaccharides. In contrast, human intelectin-1 (hItln-1, also known as Omentin-1) is a soluble lectin that binds a range of microbial sugars, including β-Dgalactofuranose (β-Galf), D-glycerol 1-phosphate, D-glycero-D-talo-oct-2-ulosonic acid (KO), and 3- deoxy-D-manno-oct-2-ulosonic acid (KDO). Though these saccharides differ dramatically in structure, they share a common feature—an exocyclic vicinal diol. How and whether such a small fragment is sufficient for recognition was unclear. We tested several glycans with this epitope and found that L-glycero-α-Dmanno- heptose and D-glycero-α-D-manno-heptose possess the critical diol motif yet bind weakly. To better understand hItln-1 recognition, we determined the structure of the hItln-1·KO complex using X-ray crystallography, and our 1.59-Å resolution structure enabled unambiguous assignment of the bound KO conformation. This carbohydrate conformation was present in >97% of the KDO/KO structures in the Protein Data Bank. Bioinformatic analysis revealed that KO and KDO adopt a common conformation, while heptoses prefer different conformers. The preferred conformers of KO and KDO favor hItln-1 engagement, but those of the heptoses do not. Natural bond orbital (NBO) calculations suggest these observed conformations, including the side chain orientations, are stabilized by not only steric but also stereoelectronic effects. Thus, our data highlight a role for stereoelectronic effects in dictating the specificity of glycan recognition by proteins. Finally, our finding that hItln-1 avoids binding prevalent glycans with a terminal 1,2 diol (e.g., NeuAc, and L-glycero-α-D-manno-heptose) suggests the lectin has evolved to recognize distinct bacterial species

Palladium–Protein Oxidative Addition Complexes by Amine-Selective Acylation

© Palladium oxidative addition complexes (OACs) are traditionally accessed by treating an aryl halide-containing substrate with a palladium(0) source. Here, a new strategy to selectively prepare stable OACs from amino groups on native proteins is presented. The approach relies on an amine-selective acylation reaction that occurs without modification of a preformed palladium(II)-aryl group. Once transferred onto a protein substrate, the palladium(II)-aryl group facilitates conjugation by undergoing reaction with a second, cysteine-containing protein. This operationally simple method is applicable to native, nonengineered enzymes as well as antibodies and is carried out in an aqueous setting and open to air. The resulting Pd-protein OACs are stable, storable reagents that retain biological activity and can be used to achieve protein-protein cross-coupling at nanomolar concentrations within hours

Stereoelectronic Effects Impact Glycan Recognition

Copyright © 2020 American Chemical Society. Recognition of distinct glycans is central to biology, and lectins mediate this function. Lectin glycan preferences are usually centered on specific monosaccharides. In contrast, human intelectin-1 (hItln-1, also known as Omentin-1) is a soluble lectin that binds a range of microbial sugars, including β-d-galactofuranose (β-Galf), d-glycerol 1-phosphate, d-glycero-d-talo-oct-2-ulosonic acid (KO), and 3-deoxy-d-manno-oct-2-ulosonic acid (KDO). Though these saccharides differ dramatically in structure, they share a common feature - an exocyclic vicinal diol. How and whether such a small fragment is sufficient for recognition was unclear. We tested several glycans with this epitope and found that l-glycero-α-d-manno-heptose and d-glycero-α-d-manno-heptose possess the critical diol motif yet bind weakly. To better understand hItln-1 recognition, we determined the structure of the hItln-1·KO complex using X-ray crystallography, and our 1.59 Å resolution structure enabled unambiguous assignment of the bound KO conformation. This carbohydrate conformation was present in >97% of the KDO/KO structures in the Protein Data Bank. Bioinformatic analysis revealed that KO and KDO adopt a common conformation, while heptoses prefer different conformers. The preferred conformers of KO and KDO favor hItln-1 engagement, but those of the heptoses do not. Natural bond orbital (NBO) calculations suggest these observed conformations, including the side chain orientations, are stabilized by not only steric but also stereoelectronic effects. Thus, our data highlight a role for stereoelectronic effects in dictating the specificity of glycan recognition by proteins. Finally, our finding that hItln-1 avoids binding prevalent glycans with a terminal 1,2-diol (e.g., N-acetyl-neuraminic acid and l-glycero-α-d-manno-heptose) suggests the lectin has evolved to recognize distinct bacterial species