Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit

In order to use a Hemoglobin Based Oxygen Carrier as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the hemoglobin molecule to prevent rapid renal clearance. A common method uses maleimide PEGylation of sulfhydryls created by the reaction of 2-iminothiolane at surface lysines. However, this creates highly heterogenous mixtures of molecules. We recently engineered a hemoglobin with a single novel, reactive cysteine residue on the surface of the alpha subunit creating a single PEGylation site (βCys93Ala/αAla19Cys). This enabled homogenous PEGylation by maleimide-PEG with >80% efficiency and no discernible effect on protein function. However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions and cross-conjugation to endogenous thiol species in vivo. We therefore compared our maleimide-PEG adduct with one created using a mono-sulfone-PEG less susceptible to deconjugation. Mono-sulfone-PEG underwent reaction at αAla19Cys hemoglobin with > 80% efficiency, although some side reactions were observed at higher PEG:hemoglobin ratios; the adduct bound oxygen with similar affinity and cooperativity as wild type hemoglobin. When directly compared to maleimide-PEG, the mono-sulfone-PEG adduct was significantly more stable when incubated at 37°C for seven days in the presence of 1 mM reduced glutathione. Hemoglobin treated with mono-sulfone-PEG retained > 90% of its conjugation, whereas for maleimide-PEG < 70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation is certainly stable enough for acute therapeutic use as an oxygen therapeutic, for pharmaceuticals intended for longer vascular retention (weeks-months), reagents such as mono-sulfone-PEG may be more appropriate

Engineering hemoglobin to enable homogenous PEGylation without modifying protein functionality

In order to infuse hemoglobin into the vasculature as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the molecule to enhance vascular retention. This aim can be achieved by PEGylation. However, using non-specific conjugation methods creates heterogenous mixtures and alters protein function. Site-specific PEGylation at the naturally reactive thiol on human hemoglobin (βCys93) alters hemoglobin oxygen binding affinity and increases its autooxidation rate. In order to avoid this issue, new reactive thiol residues were therefore engineered at sites distant to the heme group and the α/β dimer/dimer interface. The two mutants were βCys93Ala/αAla19Cys and βCys93Ala/βAla13Cys. Gel electrophoresis, size exclusion chromatography and mass spectrometry revealed efficient PEGylation at both αAla19Cys and βAla13Cys, with over 80% of the thiols PEGylated in the case of αAla19Cys. For both mutants there was no significant effect on the oxygen affinity or the cooperativity of oxygen binding. PEGylation at αAla19Cys had the additional benefit of decreasing the rates of autoxidation and heme release, properties that have been considered contributory factors to the adverse clinical side effects exhibited by previous hemoglobin based oxygen carriers. PEGylation at αAla19Cys may therefore be a useful component of future clinical products

Aptamers that recognize drug-resistant HIV-1 reverse transcriptase

Drug-resistant variants of HIV-1 reverse transcriptase (RT) are also known to be resistant to anti-RT RNA aptamers. In order to be able to develop diagnostics and therapies that can focus on otherwise drug-resistant viruses, we have isolated two aptamers against a well-known, drug-resistant HIV-1 RT, Mutant 3 (M3) from the multidrug-resistant HIV-1 RT panel. One aptamer, M302, bound M3 but showed no significant affinity for wild-type (WT) HIV-1 RT, while another aptamer, 12.01, bound to both M3 and WT HIV-1 RTs. In contrast to all previously selected anti-RT aptamers, neither of these aptamers showed observable inhibition of either polymerase or RNase H activities. Aptamers M302 and 12.01 competed with one another for binding to M3, but they did not compete with a pseudoknot aptamer for binding to the template/primer cleft of WT HIV-1 RT. These results represent the surprising identification of an additional RNA-binding epitope on the surface of HIV-1 RT. M3 and WT HIV-1 RTs could be distinguished using an aptamer-based microarray. By probing protein conformation as a correlate to drug resistance we introduce an additional and useful measure for determining HIV-1 drug resistance

Color adjectives, standards, and thresholds: an experimental investigation

Are color adjectives (“red”, “green”, etc.) relative adjectives or absolute adjectives? Existing theories of the meaning of color adjectives attempt to answer that question using informal (“armchair”) judgments. The informal judgments of theorists conflict: it has been proposed that color adjectives are absolute with standards anchored at the minimum degree on the scale, that they are absolute but have near- midpoint standards, and that they are relative. In this paper we report two experiments, one based on entailment patterns and one based on presupposition accommodation, that investigate the meaning of scalar adjectives. We find evidence confirming the existence of subgroups of the population who operate with different standards for color adjectives. The evidence of interpersonal variation in where standards are located on the relevant scale and how those standards can be adjusted indicates that the existing theories of the meaning of color adjectives are at best only partially correct. We also find evidence that paradigmatic relative adjectives (“tall”, “wide”) behave in ways that are not predicted by the standard theory of scalar adjectives. We discuss several different possible explanations for this unexpected behavior. We conclude by discussing the relevance of our findings for philosophical debates about the nature and extent of semantically encoded context sensitivity in which color adjectives have played a key role

Review of the Palaearctic species of Ismaridae Thomson, 1858 (Hymenoptera: Diaprioidea)

This is an open access article, available to all readers online, published under a Creative Commons BY-NC-ND license: https://creativecommons.org/licenses/by-nc-nd/3.0/. The attached file is the published version of the article

Evidence Use and the Institutions of the State: The Role of Parliament and the Judiciary

This chapter explores the role of parliaments and the judiciary in shaping evidence use in health policy making. Most analyses of the role of scientific evidence focus on the executive, i.e. national governments and ministries of health, as the key state actors in health policy and health system governance. This chapter shifts attention to the other two powers within the state, the legislative and the judiciary. Using the examples analysed in this book the chapter examines how parliaments can use evidence to inform legislative processes and to hold governments to account, although there are substantial differences between countries and political systems. However, there was little suggestion that such approaches were undertaken systematically. In cases in which policies are brought to court, judges may have to deal with scientific evidence within a country’s legal and constitutional framework, again with significant differences between national legal practices

Studying Public Health Law::Principles, Politics, and Populations as Patients

Public health law is firmly establishing itself as a crucial area of scholarly inquiry. Its vital importance has been sharply underscored following the outbreak of COVID-19, in response to which we have seen the institution of extreme legal measures—suchas the UK’s Coronavirus Act 2020—in efforts to control and contain the spread ofthe disease. The pandemic has also starkly exposed the complex nature of the regulatory challenges, nationally, internationally, and globally, to which such public health problems give rise. In approaching these, and other questions concerning the public’s health, such as non-communicable disease, public health law, as a field, brings notable distinctive features: these include a practical focus on populations, institutions, the prevention of ill health, protection of good health, and thepromotion of positive states of well-being; and concomitant critical approaches rooted in theories of social justice as contrasted with more narrow biomedical ethics. Such features make it in some senses atypical territory within the field of health law. Furthermore, the inherent role of political institutions places law conceptually within public health in a way that may be seen as distinguishable from law’s relationship with clinical medicine. This chapter explains how the broad reach and distinct features of public health require a commensurately broad approach to conceptualising public health law, and how distinct practical and theoretical features may be integrated into academic public health law. It also shows how public health law, with its distinct conceptualisations concerning ‘the body’ of medical jurisprudence, can both challenge and enrich medico-legal studies, and bring important perspectives within the broader field of health law

Management practices for control of ragwort species

The ragwort species common or tansy ragwort (Jacobaea vulgaris, formerly Senecio jacobaea), marsh ragwort (S. aquaticus), Oxford ragwort (S. squalidus) and hoary ragwort (S. erucifolius) are native in Europe, but invaded North America, Australia and New Zealand as weeds. The abundance of ragwort species is increasing in west-and central Europe. Ragwort species contain different groups of secondary plant compounds defending them against generalist herbivores, contributing to their success as weeds. They are mainly known for containing pyrrolizidine alkaloids, which are toxic to grazing cattle and other livestock causing considerable losses to agricultural revenue. Consequently, control of ragwort is obligatory by law in the UK, Ireland and Australia. Commonly used management practices to control ragwort include mechanical removal, grazing, pasture management, biological control and chemical control. In this review the biology of ragwort species is shortly described and the different management practices are discussed

The SPECTRUM Consortium: a new UK Prevention Research Partnership consortium focussed on the commercial determinants of health, the prevention of non-communicable diseases, and the reduction of health inequalities

The main causes of non-communicable diseases (NCDs), health inequalities and health inequity include consumption of unhealthy commodities such as tobacco, alcohol and/or foods high in fat, salt and/or sugar. These exposures are preventable, but the commodities involved are highly profitable. The economic interests of ‘Unhealthy Commodity Producers’ (UCPs) often conflict with health goals but their role in determining health has received insufficient attention. In order to address this gap, a new research consortium has been established. This open letter introduces the SPECTRUM (Shaping Public hEalth poliCies To Reduce ineqUalities and harM) Consortium: a multi-disciplinary group comprising researchers from 10 United Kingdom (UK) universities and overseas, and partner organisations including three national public health agencies in Great Britain (GB), five multi-agency alliances and two companies providing data and analytic support. Through eight integrated work packages, the Consortium seeks to provide an understanding of the nature of the complex systems underlying the consumption of unhealthy commodities, the role of UCPs in shaping these systems and influencing health and policy, the role of systems-level interventions, and the effectiveness of existing and emerging policies. Co-production is central to the Consortium’s approach to advance research and achieve meaningful impact and we will involve the public in the design and delivery of our research. We will also establish and sustain mutually beneficial relationships with policy makers, alongside our partners, to increase the visibility, credibility and impact of our evidence. The Consortium’s ultimate aim is to achieve meaningful health benefits for the UK population by reducing harm and inequalities from the consumption of unhealthy commodities over the next five years and beyond