Combining experiment and energy landscapes to explore anaerobic heme breakdown in multifunctional hemoproteins.

To survive, many pathogens extract heme from their host organism and break down the porphyrin scaffold to sequester the Fe2+ ion via a heme oxygenase. Recent studies have revealed that certain pathogens can anaerobically degrade heme. Our own research has shown that one such pathway proceeds via NADH-dependent heme degradation, which has been identified in a family of hemoproteins from a range of bacteria. HemS, from Yersinia enterocolitica, is the main focus of this work, along with HmuS (Yersinia pestis), ChuS (Escherichia coli) and ShuS (Shigella dysenteriae). We combine experiments, Energy Landscape Theory, and a bioinformatic investigation to place these homologues within a wider phylogenetic context. A subset of these hemoproteins are known to bind certain DNA promoter regions, suggesting not only that they can catalytically degrade heme, but that they are also involved in transcriptional modulation responding to heme flux. Many of the bacterial species responsible for these hemoproteins (including those that produce HemS, ChuS and ShuS) are known to specifically target oxygen-depleted regions of the gastrointestinal tract. A deeper understanding of anaerobic heme breakdown processes exploited by these pathogens could therefore prove useful in the development of future strategies for disease prevention.

The attentional cost of comparisons: Evidence for a general comparison induced delay.

The current work aimed to uncover the pattern of attention given to external comparison standards when engaged in social judgments. In a series of 5 experiments (N = 463), a Modified Spatial Cueing Task provided evidence for a general Comparison Induced Delay (CID), but found no signs of visuospatial attention (Pilot, Study 1 & 2). However, the CID did not occur if cues did not remain visually available throughout the trials (Study 3 & 4). Heterogeneity in results prompted the use of a single-paper meta-analysis including all secondary studies. A consistent CID effect was found across studies when standards remained visually available (K = 5), but not when they were masked (K = 2). No direct signs of visuospatial attentional bias were found. These results suggest that the attentional cost of engaging with external comparisons is mainly cognitive in nature, although a minor reoccurring visual component could not be excluded.

A Multilab Replication of the Ego Depletion Effect.

There is an active debate regarding whether the ego depletion effect is real. A recent preregistered experiment with the Stroop task as the depleting task and the antisaccade task as the outcome task found a medium-level effect size. In the current research, we conducted a preregistered multilab replication of that experiment. Data from 12 labs across the globe (N = 1,775) revealed a small and significant ego depletion effect, d = 0.10. After excluding participants who might have responded randomly during the outcome task, the effect size increased to d = 0.16. By adding an informative, unbiased data point to the literature, our findings contribute to clarifying the existence, size, and generality of ego depletion.

Controlled Ligand Exchange Between Ruthenium Organometallic Cofactor Precursors and a Naïve Protein Scaffold Generates Artificial Metalloenzymes Catalysing Transfer Hydrogenation.

Many natural metalloenzymes assemble from proteins and biosynthesised complexes, generating potent catalysts by changing metal coordination. Here we adopt the same strategy to generate artificial metalloenzymes (ArMs) using ligand exchange to unmask catalytic activity. By systematically testing RuII (η6 -arene)(bipyridine) complexes designed to facilitate the displacement of functionalised bipyridines, we develop a fast and robust procedure for generating new enzymes via ligand exchange in a protein that has not evolved to bind such a complex. The resulting metal cofactors form peptidic coordination bonds but also retain a non-biological ligand. Tandem mass spectrometry and 19 F NMR spectroscopy were used to characterise the organometallic cofactors and identify the protein-derived ligands. By introduction of ruthenium cofactors into a 4-helical bundle, transfer hydrogenation catalysts were generated that displayed a 35-fold rate increase when compared to the respective small molecule reaction in solution.

The use of the independent sector in providing NHS services during the Covid-19 outbreak; two hospitals experience.

This paper reviews the activity undertaken between a teaching hospital and its adjacent Independent Hospital and its implementation under the Independent Sector Provider Contract between NHSE and the Independent Sector. RESULTS: From the instigation of the NHSE contract with the Independent Sector up until 28th June 2020 The Norfolk and Norwich University NHS Trust (NNUH) delivered 9016 episodes of care including 576 surgical episodes at its nearby Independent Hospital. During the time that a seven day household isolation period was required, no patients from the 31 tested postoperatively were recorded as testing positive for Covid-19. In the month after moving to a mandatory 14 day period of household isolation, 29 patients had their surgery postponed as they were unable to comply with the required period of isolation. CONCLUSION: Working cooperatively with the independent sector can deliver significant additional capacity for the NHS. Fourteen days household isolation may impact on a patient's decision to have surgery, despite, in some cases, that surgery being time-sensitive. The recommendation from NICE reducing the length of isolation largely reversed this impact.

To What Extent Do Erotic Images Elicit Visuospatial versus Cognitive Attentional Processes? Consistent Support for a (Non-Spatial) Sexual Content-Induced Delay.

It is almost a cultural truism that erotic images attract our attention, presumably because paying attention to erotic stimuli provided our ancestors with mating benefits. Attention, however, can be narrowly defined as visuospatial attention (keeping such stimuli in view) or more broadly as cognitive attention (such stimuli taking up one's thoughts). We present four independent studies aiming to test the extent to which erotic images have priority in capturing visuospatial versus cognitive attention. Whereas the former would show in quicker reactions to stimuli presented in locations where erotic images appeared previously, the latter causes delayed responding after erotic images, independent of their location). To this end, we specifically modified spatial cueing tasks to disentangle visuospatial attention capture from general sexual content-induced delay (SCID) effects-a major drawback in the previous literature. Consistently across all studies (total N = 399), we found no evidence in support of visuospatial attention capture but reliably observed an unspecific delay of responding for trials in which erotic images appeared (irrespective of cue location). This SCID is equally large for heterosexual men and women and reliably associated with their self-reported sexual excitability.

VENM: An Algorithm to Accurately Calculate Neutral Slopes and Gradients.

Mesoscale eddies stir along the neutral plane, and the resulting neutral diffusion is a fundamental aspect of subgrid-scale tracer transport in ocean models. Calculating neutral diffusion traditionally involves calculating neutral slopes and three-dimensional tracer gradients. The calculation of the neutral slope traditionally occurs by computing the ratio of the horizontal to vertical locally referenced potential density derivative. However, this approach is problematic in regions of weak vertical stratification, prompting the use of a variety of ad hoc regularization methods that can lead to rather nonphysical dependencies for the resulting neutral tracer gradients. Here we use a VErtical Non-local Method "VENM," a search algorithm that requires no ad hoc regularization and significantly improves the numerical accuracy of calculating neutral slopes, neutral tracer gradients, and associated neutral diffusive fluxes. We compare and contrast VENM against a more traditional method, using an independent objective neutrality condition combined with estimates of spurious diffusion, heat transport, and water mass transformation rates. VENM is more accurate, both physically and numerically, and should form the basis for future efforts involving neutral diffusion calculations from observations and possibly numerical model simulations.

Use of a fluorinated probe to quantitatively monitor amino acid binding preferences of ruthenium(ii) arene complexes.

In order to address outstanding questions about ruthenium complexes in complex biological solutions, 19F NMR spectroscopy was used to follow the binding preferences between fluorinated RuII(η6-arene)(bipyridine) complexes and protected amino acids and glutathione. Reporting what ruthenium compounds bind to in complex environments has so far been restricted to relatively qualitative methods, such as mass spectrometry and X-ray spectroscopic methods; however, quantitative information on the species present in the solution phase cannot be inferred from these techniques. Furthermore, using 1H NMR, in water, to distinguish and monitor a number of different complex RuII(η6-arene) adducts forming is challenging. Incorporating an NMR active heteroatom into ruthenium organometallic complexes provides a quantitative, diagnostic 'fingerprint' to track solution-phase behaviour and allow for unambiguous assignment of any given adduct. The resulting 19F NMR spectra show for the first time the varied, dynamic behaviour of organoruthenium compounds when exposed to simple biomolecules in complex mixtures. The rates of formation of the different observed species are dramatically influenced by the electronic properties at the metal, even in a closely related series of complexes in which only the electron-donating properties of the arene ligand are altered. Preference for cysteine binding is absolute: the first quantitative solution-phase evidence of such behaviour.

Revisiting Health Regionalization in Canada.

Twenty years ago, many of Canada's provinces began to introduce regional health authorities to address problems with their health care systems. With this action, the provinces sought to achieve advances in community decision-making, the integration of health services, and the provision of care in the home and community. The authorities were also to help restrict health care costs. An assessment of the authorities indicates, however, that over the past two decades they have been unable to meet their objectives. Community representatives continue to play little role in determining the appropriate health services for their regions. Gains have been made towards integrating health services, but the plan for a near seamless set of health services has not been realized. Funding for health services remains focused on hospital and physician care, and health care expenditures have until very recently been little affected by regional authorities. This disappointing performance has caused some provinces to abandon their regional authorities, but this article argues that the provision of greater autonomy and a better public appreciation of their role and potential may lead to more successful regional authorities. Accordingly, the objective of this article is to reveal the shortcomings of regional health authorities in Canada while at the same time arguing that changes can be made to increase the chances of more workable authorities.

Evaluating handoffs in the context of a communication framework.

BACKGROUND: The implementation of mandated restrictions in resident duty hours has led to increased handoffs for patient care and thus more opportunities for errors during transitions of care. Much of the current handoff literature is empiric, with experts recommending the study of handoffs within an established framework. METHODS: A prospective, single-institution study was conducted evaluating the process of handoffs for the care of surgical patients in the context of a published communication framework. Evaluation tools for the source, receiver, and observer were developed to identify factors impacting the handoff process, and inter-rater correlations were assessed. Data analysis was generated with Pearson/Spearman correlations and multivariate linear regressions. Rater consistency was assessed with intraclass correlations. RESULTS: A total of 126 handoffs were observed. Evaluations were completed by 1 observer (N = 126), 2 observers (N = 23), 2 receivers (N = 39), 1 receiver (N = 82), and 1 source (N = 78). An average (±standard deviation) service handoff included 9.2 (±4.6) patients, lasted 9.1 (±5.4) minutes, and had 4.7 (±3.4) distractions recorded by the observer. The source and receiver(s) recognized distractions in >67% of handoffs, with the most common internal and external distractions being fatigue (60% of handoffs) and extraneous staff entering/exiting the room (31%), respectively. Teams with more patients spent less time per individual patient handoff (r = -0.298; P = .001). Statistically significant intraclass correlations (P ≤ .05) were moderate between observers (r ≥ 0.4) but not receivers (r < 0.4). Intraclass correlation values between different types of raters were inconsistent (P > .05). The quality of the handoff process was affected negatively by presence of active electronic devices (ß = -0.565; P = .005), number of teaching discussions (ß = -0.417; P = .048), and a sense of hierarchy between source and receiver (ß = -0.309; P = .002). CONCLUSION: Studying the handoff process within an established framework highlights factors that impair communication. Internal and external distractions are common during handoffs and along with the working relationship between the source and receiver impact the quality of the handoff process. This information allows further study and targeted interventions of the handoff process to improve overall effectiveness and patient safety of the handoff.

How to make a porphyrin flip: dynamics of asymmetric porphyrin oligomers.

We present the first predictions of meso-aryl flipping pathways in porphyrin oligomers. In the context of cyclic oligoporphyrins this flipping results in a paddle rotation of each porphyrin monomer in the oligomeric ring. If the monomer porphyrin units are asymmetric, this flipping will have consequences for their supramolecular behaviour. Desymmetrisation of synthetic porphyrins leads to synthetic challenges, and hence these species are not as well studied as the more accessible, symmetric counterparts. We have both simulated and synthesized novel, desymmetrised monomeric and cyclic trimeric porphyrins and we predict that the flipping barrier for a porphyrin monomer within the trimer is 36.7 kJ mol(-1) higher than that for meso-aryl flipping in the monomer. The flipping rates estimated from Variable temperature NMR data are consistent with these results. We have also carried out a systematic investigation of how porphyrinic substituents will affect the dynamics, revealing that adding steric bulk in the right place can facilitate meso-aryl flipping. While supramolecular chemistry often focuses on highly symmetric assemblies, evolution can break molecular symmetry in subtle ways, leading to many pseudosymmetric assemblies in biology, especially protein-porphyrinic complexes that are important for energy harvesting and electron transport systems. The dynamic behaviour we have characterized can be critical for the design and function of these molecules, and hence our results will help inform future efforts in the synthesis of asymmetric porphyrinic assemblies that interact with biomolecules.

Theory and practice: bulk synthesis of C3B and its H2- and Li-storage capacity.

Previous theoretical studies of C3B have suggested that boron-doped graphite is a promising H2- and Li-storage material, with large maximum capacities. These characteristics could lead to exciting applications as a lightweight H2-storage material for automotive engines and as an anode in a new generation of batteries. However, for these applications to be realized a synthetic route to bulk C3B must be developed. Here we show the thermolysis of a single-source precursor (1,3-(BBr2)2C6H4) to produce graphitic C3B, thus allowing the characteristics of this elusive material to be tested for the first time. C3B was found to be compositionally uniform but turbostratically disordered. Contrary to theoretical expectations, the H2- and Li-storage capacities are lower than anticipated, results that can partially be explained by the disordered nature of the material. This work suggests that to model the properties of graphitic materials more realistically, the possibility of disorder must be considered.

Investigating the mechanisms of amylolysis of starch granules by solution-state NMR.

Starch is a prominent component of the human diet and is hydrolyzed by α-amylase post-ingestion. Probing the mechanism of this process has proven challenging, due to the intrinsic heterogeneity of individual starch granules. By means of solution-state NMR, we demonstrate that flexible polysaccharide chains protruding from the solvent-exposed surfaces of waxy rice starch granules are highly mobile and that during hydrothermal treatment, when the granules swell, the number of flexible residues on the exposed surfaces increases by a factor of 15. Moreover, we show that these flexible chains are the primary substrates for α-amylase, being cleaved in the initial stages of hydrolysis. These findings allow us to conclude that the quantity of flexible α-glucan chains protruding from the granule surface will greatly influence the rate of energy acquisition from digestion of starch.

Selective lability of ruthenium(II) arene amino acid complexes.

A series of organometallic complexes of the form [(PhH)Ru(amino acid)](+) have been synthesized using amino acids able to act as tridentate ligands. The straightforward syntheses gave enantiomerically pure complexes with two stereogenic centers due to the enantiopurity of the chelating ligands. Complexes were characterized in the solid-state and/or solution-state where the stability of the complex allowed. The propensity toward labilization of the coordinatively saturated complexes was investigated. The links between complex stability and structural features are very subtle. Nonetheless, H/D exchange rates of coordinated amino groups reveal more significant differences in reactivity linked to metallocycle ring size resulting in decreasing stability of the metallocycle as the amino acid side-chain length increases. The behavior of these systems in acid is unusual, apparently labilizing the carboxylate residue of the amino acid. This acid-catalyzed hemilability in an organometallic is relevant to the use of Ru(II) arenes in medicinal contexts due to the relatively low pH of cancerous cells.

Folates are potential ligands for ruthenium compounds in vivo.

Under physiologically relevant conditions, cis-bis(2,2'-bipyridine)dichlororuthenium(II), [cis-Ru(2,2'-bipy)2Cl2] was observed to bind to folic acid via replacement of the two chloride ligands. This binding was shown to be pH dependent and afforded diastereomers, the structures of which were determined by 1- and 2D NMR spectroscopic techniques. We propose that when studying the cytotoxicity of labile ruthenium complexes in cells, folate coordination should be considered.

Local frustration determines molecular and macroscopic helix structures.

Decorative domains force amyloid fibers to adopt spiral ribbon morphologies, as opposed to the more common twisted ribbon. We model the effect of decorating domains as a perturbation to the relative orientation of ß strands in a bilayered extended ß-sheet. The model consists of minimal energy assemblies of rigid building blocks containing two anisotropic interacting ellipsoids. The relative orientation of the ellipsoids dictates the morphology of the resulting assembly. Amyloid structures derived from experiment are consistent with our model, and we use magnets to demonstrate that the frustration principle is scale and system independent. In contrast to other models of amyloid, our model isolates the effect of frustration from the fundamental interactions between building blocks to reveal the frustration rather than dependence of morphology on the physical interactions. Consequently, amyloid is viewed as a discrete molecular version of the more general macroscopic frustrated bilayer that is exemplified by Bauhinia seedpods. The model supports the idea that the interactions arising from an arbitrary peptide sequence can support an amyloid structure if a bilayer can form first, which suggests that supplementary protein sequences, such as chaperones or decorative domains, could play a significant role in stabilizing such bilayers and therefore in selecting morphology during nucleation. Our model provides a foundation for exploring the effects of frustration on higher-order superstructural polymorphic assemblies that may exhibit complex functional behavior. Two outstanding examples are the systematic kinking of decorated fibers and the nested frustration of the Bauhinia seedpod.

Can a computer-generated voice be sincere? A case study combining music and synthetic speech.

This article explores enhancing sincerity, honesty, or truthfulness in computer-generated synthetic speech by accompanying it with music. Sincerity is important if we are to respond positively to any voice, whether human or artificial. What is sincerity in the artificial disembodied voice? Studies in musical expression and performance may illuminate aspects of the 'musically spoken' or sung voice in rendering deeper levels of expression that may include sincerity. We consider one response to this notion in an especially composed melodrama (music accompanying a (synthetic) spoken voice) designed to convey sincerity.

Structural basis for efficient chromophore communication and energy transfer in a constructed didomain protein scaffold.

The construction of useful functional biomolecular components not currently part of the natural repertoire is central to synthetic biology. A new light-capturing ultra-high-efficiency energy transfer protein scaffold has been constructed by coupling the chromophore centers of two normally unrelated proteins: the autofluorescent protein enhanced green fluorescent protein (EGFP) and the heme-binding electron transfer protein cytochrome b(562) (cyt b(562)). Using a combinatorial domain insertion strategy, a variant was isolated in which resonance energy transfer from the donor EGFP to the acceptor cyt b(562) was close to 100% as evident by virtually full fluorescence quenching on heme binding. The fluorescence signal of the variant was also sensitive to the reactive oxygen species H(2)O(2), with high signal gain observed due to the release of heme. The structure of oxidized holoprotein, determined to 2.75 Å resolution, revealed that the two domains were arranged side-by-side in a V-shape conformation, generating an interchromophore distance of ~17 Å (14 Å edge-to-edge). Critical to domain arrangement is the formation of a molecular pivot point between the two domains as a result of different linker sequence lengths at each domain junction and formation of a predominantly polar interdomain interaction surface. The retrospective structural analysis has provided an explanation for the basis of the observed highly efficient energy transfer through chromophore arrangement in the directly evolved protein scaffold and provides an insight into the molecular principles by which to design new proteins with coupled functions.

Continued surprises in the cytochrome c biogenesis story.

Cytochromes c covalently bind their heme prosthetic groups through thioether bonds between the vinyl groups of the heme and the thiols of a CXXCH motif within the protein. In Gram-negative bacteria, this process is catalyzed by the Ccm (cytochrome c maturation) proteins, also called System I. The Ccm proteins are found in the bacterial inner membrane, but some (CcmE, CcmG, CcmH, and CcmI) also have soluble functional domains on the periplasmic face of the membrane. Elucidation of the mechanisms involved in the transport and relay of heme and the apocytochrome from the bacterial cytosol into the periplasm, and their subsequent reaction, has proved challenging due to the fact that most of the proteins involved are membrane-associated, but recent progress in understanding some key components has thrown up some surprises. In this Review, we discuss advances in our understanding of this process arising from a substrate's point of view and from recent structural information about individual components.