Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12)

It has been known for the past 20 years that two pathways exist in nature for the de novo biosynthesis of the coenzyme form of vitamin B12, adenosylcobalamin, representing aerobic and anaerobic routes. In contrast to the aerobic pathway, the anaerobic route has remained enigmatic because many of its intermediates have proven technically challenging to isolate, because of their inherent instability. However, by studying the anaerobic cobalamin biosynthetic pathway in Bacillus megaterium and using homologously overproduced enzymes, it has been possible to isolate all of the intermediates between uroporphyrinogen III and cobyrinic acid. Consequently, it has been possible to detail the activities of purified cobinamide biosynthesis (Cbi) proteins CbiF, CbiG, CbiD, CbiJ, CbiET, and CbiC, as well as show the direct in vitro conversion of 5-aminolevulinic acid into cobyrinic acid using a mixture of 14 purified enzymes. This approach has resulted in the isolation of the long sought intermediates, cobalt-precorrin-6A and -6B and cobalt-precorrin-8. EPR, in particular, has proven an effective technique in following these transformations with the cobalt(II) paramagnetic electron in the dyz orbital, rather than the typical dz2. This result has allowed us to speculate that the metal ion plays an unexpected role in assisting the interconversion of pathway intermediates. By determining a function for all of the pathway enzymes, we complete the tool set for cobalamin biosynthesis and pave the way for not only enhancing cobalamin production, but also design of cobalamin derivatives through their combinatorial use and modification

Professionalism and person-centredness: developing a practice based approach to leadership within NHS maternity services in the UK

This paper, based on data taken from in-depth interviews with senior midwives and obstetricians and conducted as part of a critical ethnographic study, argues for a greater appreciation of person-centred, value-led midwifery practice. The paper begins with a discussion of the way midwifery practice is shaped by encoded and embodied knowledge. The paper subsequently focuses on an emergent practice based leadership using an adapted Aristotelian conceptual framework derived from MacIntyre (2007). Professional dissonance is highlighted as a difficulty experienced by repositioned managers who are also expected to be leaders in their field. Using data gathered from in-depth interviews it is contended that establishing person-centred care might be better achieved through the development of practice based leadership (rather than solely by adherence to organisational requirements). This type of leadership could potentially nurture a professional environment that promotes qualities, such as agency, commitment and high levels of competence among midwives. Such leadership is central to UK government priorities and is applicable to a global practice development agenda

Live Reality Television: care structures within the production and reception of talent shows

This article focuses on production and reception practices for live reality television, using critical theory and empirical research to question how producers and audiences co-create and limit live experiences. The concept of care structures is used to make visible hidden labour in the creation of mood, in particular audiences as participants in the management of live experiences. In the case of Got to Dance there was a play off between the value and meaning of the live events as a temporary experience captured by ratings and social media, and the more enduring collective-social experience of this reality series over time

Tourism destination modelling: building a sustainable planning tool for Australian tourism destinations

The Ningaloo Destination Model is a tourism planning tool for the Ningaloo Coast region of Western Australia that assesses the economic, social and environmental impacts of different planning decisions and events. This report describes the features of the tourism destination model, and analyses its application in the region and to other parts of Australia. Destination modelling integrates a number of research methodologies developed through past STCRC projects (on visitor spending and characteristics, social impacts and economic impacts), secondary data and ecological research. The key to this process is a model development technique that uses scenario planning methodologies to facilitate stakeholder engagement and conceptual modelling techniques to facilitate research integration. The report describes the methodologies used for model development and for data collection, provides two case studies demonstrating outputs, and explores applications of the Ningaloo Destination Model to the region, to other locations and to other sectors. The Ningaloo Destination Model provides estimates of the impacts of plans and events related to tourism in four dimensions: tourism specific; economy; social; and environmental (both resource use and ecological). These outputs are explored through two case studies: a nodal coastal development; and a large resort development. The model can be used for four broad (oftentimes overlapping) categories of assessment: operational planning and decision making for specific organisations and groups (such as local government or agencies that manage land or sea use), regional planning, participatory planning and collaborations and to assist monitoring and evaluation.The Ningaloo Destination Model will be available to the general public (in a limited format) through websites; to agencies through a desktop version; and through integration into a larger model of the region being developed by the CSIRO. Destination modelling is relevant for other tourism destinations and a process for the rapid and cost-effective application of destination modelling is feasible. While much of the data is available, a broader roll-out would require benchmarking of water, electricity and waste data, and developing a wireframe for all tourism destinations. Making destination modelling tools broadly available would significantly broaden the impacts considered in tourism planning and lead to enhancement of desirable effects of tourism development, and early mitigation of negative impacts across Australia. The techniques developed for destination modelling were also found to be applicable to other sectors

Calculating metalation in cells reveals CobW acquires Co(II) for vitamin B12 biosynthesis while related proteins prefer Zn(II)

Protein metal-occupancy (metalation) in vivo has been elusive. To address this challenge, the available free energies of metals have recently been determined from the responses of metal sensors. Here, we use these free energy values to develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. We use the calculator to understand the function and mechanism of GTPase CobW, a predicted CoII-chaperone for vitamin B12. Upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator also reveals that related GTPases with comparable ZnII affinities to CobW, preferentially acquire ZnII due to their relatively weaker CoII affinities. The calculator is made available here for use with other proteins

Replacement of the Cobalt Center of Vitamin B 12 by Nickel: Nibalamin and Nibyric Acid Prepared from Metal‐Free B 12 Ligands Hydrogenobalamin and Hydrogenobyric Acid

The (formal) replacement of Co in cobalamin (Cbl) by NiII generates nibalamin (Nibl), a new transition‐metal analogue of vitamin B12. Described here is Nibl, synthesized by incorporation of a NiII ion into the metal‐free B12 ligand hydrogenobalamin (Hbl), itself prepared from hydrogenobyric acid (Hby). The related NiII corrin nibyric acid (Niby) was similarly synthesized from Hby, the metal‐free cobyric acid ligand. The solution structures of Hbl, and Niby and Nibl, were characterized by spectroscopic studies. Hbl features two inner protons bound at N2 and N4 of the corrin ligand, as discovered in Hby. X‐ray analysis of Niby shows the structural adaptation of the corrin ligand to NiII ions and the coordination behavior of NiII. The diamagnetic Niby and Nibl, and corresponding isoelectronic CoI corrins, were deduced to be isostructural. Nibl is a structural mimic of four‐coordinate base‐off Cbls, as verified by its ability to act as a strong inhibitor of bacterial adenosyltransferase

Total Synthesis, Structure, and Biological Activity of Adenosylrhodibalamin, the Non-Natural Rhodium Homologue of Coenzyme B12.

B12 is unique among the vitamins as it is biosynthesized only by certain prokaryotes. The complexity of its synthesis relates to its distinctive cobalt corrin structure, which is essential for B12 biochemistry and renders coenzyme B12 (AdoCbl) so intriguingly suitable for enzymatic radical reactions. However, why is cobalt so fit for its role in B12‐dependent enzymes? To address this question, we considered the substitution of cobalt in AdoCbl with rhodium to generate the rhodium analogue 5′‐deoxy‐5′‐adenosylrhodibalamin (AdoRbl). AdoRbl was prepared by de novo total synthesis involving both biological and chemical steps. AdoRbl was found to be inactive in vivo in microbial bioassays for methionine synthase and acted as an in vitro inhibitor of an AdoCbl‐dependent diol dehydratase. Solution NMR studies of AdoRbl revealed a structure similar to that of AdoCbl. However, the crystal structure of AdoRbl revealed a conspicuously better fit of the corrin ligand for RhIII than for CoIII, challenging the current views concerning the evolution of corrins

Stakeholder perspectives on barriers and enablers to recruiting anxious children undergoing day surgery under general anaesthetic : a qualitative internal pilot study of the MAGIC randomised controlled trial

Background The ‘Melatonin for Anxiety prior to General anaesthesia In Children’ (MAGIC) trial was designed to compare midazolam and melatonin as pre-medications for anxious children (aged five to fourteen), undergoing day-case surgical procedures under general anaesthesia. Low recruitment is a challenge for many trials, particularly paediatric trials and those in ‘emergency’ settings. A qualitative study as part of MAGIC aimed to gather stakeholder perspectives on barriers and enablers to recruitment. Methods Sixteen stakeholders from six sites participated in semi-structured interviews about their experiences of setting up the MAGIC trial and recruiting patients as part of the internal pilot. Data was analysed using framework analysis. Results Participants identified barriers and enablers to recruitment. Barriers and enablers related to the study, participants, the population of anxious children, practitioners, collaboration with other health professionals, ethics, specific settings and the context of surgical day units and the wider health system. Attempting to recruit anxious children from a surgical day unit is particularly challenging for several reasons. Issues include the practicalities of dealing with a child experiencing anxiety for parents/guardians; professional unwillingness to make things more difficult for families and clinicians and nurses valuing predictability within a busy and time-sensitive setting. Conclusions Multi-site RCTs face recruitment barriers relating to study-wide and site-specific factors. There are multiple barriers to recruiting anxious children due to undergo day-case surgery. Barriers across domains can interrelate and reinforce each other, reflecting challenges relating to populations and settings. For example, in the case of anxious children, parents and other health professionals are concerned about exacerbating children’s anxiety prior to surgery. They may look for ways to keep things predictable and avoid the uncertainty of an RCT. Pre-trial engagement work could help address concerns among collaborating health professionals. Using rapid ethnography during set-up or an internal pilot to focus on how the protocol will be or has been operationalised in practice may help identify issues. Allowing time to reflect on the findings of internal pilots and implement necessary changes could facilitate higher recruitment during the main phase of a trial. Trial registration NIHR Trial Registration Number: ISRCTN18296119. Registered on October 01, 2019

Plasmodium falciparum hydroxymethylbilane synthase does not house any cosynthase activity within the haem biosynthetic pathway

Uroporphyrinogen III, the universal progenitor of macrocyclic, modified tetrapyrroles, is produced from aminolaevulinic acid (ALA) by a conserved pathway involving three enzymes: porphobilinogen synthase (PBGS), hydroxymethylbilane synthase (HmbS) and uroporphyrinogen III synthase (UroS). The gene encoding uroporphyrinogen III synthase has not yet been identified in Plasmodium falciparum, but it has been suggested that this activity is housed inside a bifunctional hybroxymethylbilane synthase (HmbS). Additionally, an unknown protein encoded by PF3D7_1247600 has also been predicted to possess UroS activity. In this study it is demonstrated that neither of these proteins possess UroS activity and the real UroS remains to be identified. This was demonstrated by the failure of codon-optimized genes to complement a defined Escherichia coli hemD− mutant (SASZ31) deficient in UroS activity. Furthermore, HPLC analysis of the oxidized reaction product from recombinant, purified P. falciparum HmbS showed that only uroporphyrin I could be detected (corresponding to hydroxymethylbilane production). No uroporphyrin III was detected, showing that P. falciparum HmbS does not have UroS activity and can only catalyze the formation of hydroxymethylbilane from porphobilinogen