Virtual Wards: A Rapid Adaptation to Clinical Attachments in MBChB During the COVID-19 Pandemic.

When the COVID-19 pandemic suddenly prevented medical students from attending their clinical attachments, the faculty involved in the third year of medical school (MBChB3) at the University of Glasgow created Virtual Wards. The focus of the Virtual Wards was to continue teaching of clinical reasoning remotely whilst COVID-19 restrictions were in place. Virtual Wards were mapped to the common and important presentations and conditions and provided opportunity for history-taking, clinical examination skills, requesting investigations, interpreting results, diagnosis and management. The Virtual Wards were successful, and further wards were developed the following academic year for MBChB4 students. This chapter describes the theoretical underpinnings of the Virtual Wards and the technological considerations, followed by a description of the Wards themselves. We then analyse an evaluation of the Virtual Wards and provide both a faculty and student perspective. Throughout the chapter, we provide tips for educators developing Virtual Ward environments.

Influence of combined yeast culture and enzymatically hydrolyzed yeast on in vitro ruminal fermentation in contrasting feed substrates.

BACKGROUND: Feed additives such as live yeast cultures have increasingly been used in ruminant feeds to improve animal performance and feeding efficiency. However, it is not clear how inactive combined yeast cultures affect ruminal gas production, fermentation kinetics and efficiency. Therefore, this study was done to determine the influence of incubating different substrates with a combined yeast culture + enzymatically hydrolyzed yeast (YC + EHY) on in vitro ruminal gas production, fermentation kinetics and metabolizable energy. Six contrasting substrates (Trichantera gigantea and Glircidia sepium leaves, Brachiaria hybrid (cv. Mulato II) leaf + stem and leaf only, Cynodon nlemfuensis and a commercial concentrate dairy feed) were incubated with and without YC + EHY in buffered rumen fluid and gas production measured at 2, 4, 6, 8, 10, 12, 15, 19, 24, 30, 36, 48 and 72 h post incubation. RESULTS: In vitro fermentation parameters (a, b, a + b and c) were unaffected by YC + EHY except for the lag phase in T. gigantea, which that reduced by 31.3% when it was incubated with YC + EHY. Supplementation with YC + EHY also did not affect metabolizable energy, 72 h organic matter digestibility, 24 h gas or CH4 production within substrate. However, cumulative gas and methane production at peak fermentation in the commercial concentrate feed was reduced by 20% when incubated with YC + EHY. CONCLUSION: It was concluded that YC + EHY has the potential to improve microbial colonization of T. gigantean substrates and reduce gas and methane production at peak fermentation in commercial concentrate feeds. © 2021 Society of Chemical Industry.

A Framework for Understanding Uncertainty in Seismic Risk Assessment.

A better understanding of the uncertainty that exists in models used for seismic risk assessment is critical to improving risk-based decisions pertaining to earthquake safety. Current models estimating the probability of collapse of a building do not consider comprehensively the nature and impact of uncertainty. This article presents a model framework to enhance seismic risk assessment and thus gives decisionmakers a fuller understanding of the nature and limitations of the estimates. This can help ensure that risks are not over- or underestimated and the value of acquiring accurate data is appreciated fully. The methodology presented provides a novel treatment of uncertainties in input variables, their propagation through the model, and their effect on the results. The study presents ranges of possible annual collapse probabilities for different case studies on buildings in different parts of the world, exposed to different levels of seismicity, and with different vulnerabilities. A global sensitivity analysis was conducted to determine the significance of uncertain variables. Two key outcomes are (1) that the uncertainty in ground-motion conversion equations has the largest effect on the uncertainty in the calculation of annual collapse probability; and (2) the vulnerability of a building appears to have an effect on the range of annual collapse probabilities produced, i.e., the level of uncertainty in the estimate of annual collapse probability, with less vulnerable buildings having a smaller uncertainty.

Why should we investigate the morphological disparity of plant clades?

BACKGROUND: Disparity refers to the morphological variation in a sample of taxa, and is distinct from diversity or taxonomic richness. Diversity and disparity are fundamentally decoupled; many groups attain high levels of disparity early in their evolution, while diversity is still comparatively low. Diversity may subsequently increase even in the face of static or declining disparity by increasingly fine sub-division of morphological 'design' space (morphospace). Many animal clades reached high levels of disparity early in their evolution, but there have been few comparable studies of plant clades, despite their profound ecological and evolutionary importance. This study offers a prospective and some preliminary macroevolutionary analyses. METHODS: Classical morphometric methods are most suitable when there is reasonable conservation of form, but lose traction where morphological differences become greater (e.g. in comparisons across higher taxa). Discrete character matrices offer one means to compare a greater diversity of forms. This study explores morphospaces derived from eight discrete data sets for major plant clades, and discusses their macroevolutionary implications. KEY RESULTS: Most of the plant clades in this study show initial, high levels of disparity that approach or attain the maximum levels reached subsequently. These plant clades are characterized by an initial phase of evolution during which most regions of their empirical morphospaces are colonized. Angiosperms, palms, pines and ferns show remarkably little variation in disparity through time. Conifers furnish the most marked exception, appearing at relatively low disparity in the latest Carboniferous, before expanding incrementally with the radiation of successive, tightly clustered constituent sub-clades. CONCLUSIONS: Many cladistic data sets can be repurposed for investigating the morphological disparity of plant clades through time, and offer insights that are complementary to more focused morphometric studies. The unique structural and ecological features of plants make them ideally suited to investigating intrinsic and extrinsic constraints on disparity.

Clades reach highest morphological disparity early in their evolution.

There are few putative macroevolutionary trends or rules that withstand scrutiny. Here, we test and verify the purported tendency for animal clades to reach their maximum morphological variety relatively early in their evolutionary histories (early high disparity). We present a meta-analysis of 98 metazoan clades radiating throughout the Phanerozoic. The disparity profiles of groups through time are summarized in terms of their center of gravity (CG), with values above and below 0.50 indicating top- and bottom-heaviness, respectively. Clades that terminate at one of the "big five" mass extinction events tend to have truncated trajectories, with a significantly top-heavy CG distribution overall. The remaining 63 clades show the opposite tendency, with a significantly bottom-heavy mean CG (relatively early high disparity). Resampling tests are used to identify groups with a CG significantly above or below 0.50; clades not terminating at a mass extinction are three times more likely to be significantly bottom-heavy than top-heavy. Overall, there is no clear temporal trend in disparity profile shapes from the Cambrian to the Recent, and early high disparity is the predominant pattern throughout the Phanerozoic. Our results do not allow us to distinguish between ecological and developmental explanations for this phenomenon. To the extent that ecology has a role, however, the paucity of bottom-heavy clades radiating in the immediate wake of mass extinctions suggests that early high disparity more probably results from the evolution of key apomorphies at the base of clades rather than from physical drivers or catastrophic ecospace clearing.

The Clinical Frailty Scale can be used retrospectively to assess the frailty of patients with hip fracture: a validation study.

PURPOSE: Frailty is a common clinical syndrome affecting hip fracture patients. Recognising and accurately assessing frailty status is important in clinical and research settings. The Rockwood Clinical Frailty Scale (CFS) is a commonly used instrument and demonstrates a strong correlation with mortality and length of hospital admission following hip fracture. What is not understood, however, is the validity of retrospectively assigned CFS scores in hip fracture patients. The aim of this study was to assess the validity of retrospective non-orthogeriatrician assigned CFS scores in hip fracture patients. METHODS: Hip fracture patients from a single major trauma centre were assessed and CFS scores were assigned prospectively by non-orthogeriatric clinicians (n = 57). A subset of these patients were also assigned a prospective CFS score by a specialist orthogeriatrician (n = 27). Two separate blinded observers (non-orthogeriatric clinicians) assigned CFS scores retrospectively using electronic patient records alone. Agreement and precision was examined using the Bland-Altman plot, accuracy was assessed using R2 statistic and inter-rater reliability was assessed using quadratic weighted Cohen's kappa. RESULTS: Seventy percent of the cohort were female with an average age of 83. Agreement was high between prospective non-orthogeriatrician assigned CFS scores and retrospective non-orthogeriatrician assigned CFS scores, with a low bias (0.046) and good accuracy (R2 = 73%). Good agreement was also seen in comparisons between prospective orthogeriatrician assigned CFS scores versus retrospective non-orthogeriatrician assigned scores, with a low bias (0.23) and good accuracy (R2 = 78%). Good inter-rater reliability was seen between blinded observers with a quadratic weighted Cohen's kappa of 0.76. CONCLUSIONS: Retrospective CFS scores assigned by non-orthogeriatricians are a valid means of assessing frailty status in hip fracture patients. However, our results suggest a tendency for non-orthogeriatricians to marginally overestimate frailty status when assigning CFS scores retrospectively.

Multicenter, prospective clinical evaluation of respiratory samples from subjects at risk for Pneumocystis jirovecii infection by use of a commercial real-time PCR assay.

Pneumocystis jirovecii pneumonia (PCP) is a common opportunistic infection. Microscopic diagnosis, including diagnosis using the Merifluor-Pneumocystis direct fluorescent antigen (MP-DFA) test, has limitations. Real-time PCR may assist in diagnosis, but no commercially validated real-time PCR assay has been available to date. MycAssay Pneumocystis is a commercial assay that targets the P. jirovecii mitochondrial large subunit (analytical detection limit, ≤ 3.5 copies/µl of sample). A multicenter trial recruited 110 subjects: 54 with transplants (40 with lung transplants), 32 with nonmalignant conditions, 13 with leukemia, and 11 with solid tumors; 9 were HIV positive. A total of 110 respiratory samples (92% of which were bronchoalveolar lavage [BAL] specimens) were analyzed by PCR. Performance was characterized relative to investigator-determined clinical diagnosis of PCP (including local diagnostic tests), and PCR results were compared with MP-DFA test results for 83 subjects. Thirteen of 14 subjects with PCP and 9/96 without PCP (including 5 undergoing BAL surveillance after lung transplantation) had positive PCR results; sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) were 93%, 91%, 59%, and 99%, respectively. Fourteen of 83 subjects for whom PCR and MP-DFA test results were available had PCP; PCR sensitivity, specificity, PPV, and NPV were 93%, 90%, 65%, and 98%, respectively, and MP-DFA test sensitivity, specificity, PPV, and NPV were 93%, 100%, 100%, and 98%. Of the 9 PCR-positive subjects without PCP, 1 later developed PCP. The PCR diagnostic assay compares well with clinical diagnosis using nonmolecular methods. Additional positive results compared with the MP-DFA test may reflect low-level infection or colonization.

The influence of different aspects of grouse moorland management on nontarget bird assemblages.

Conflict between stakeholders with opposing interests can hamper biodiversity conservation. When conflicts become entrenched, evidence from applied ecology can reveal new ways forward for their management. In particular, where disagreement exists over the efficacy or ethics of management actions, research clarifying the uncertain impacts of management on wildlife can move debates forwards to conciliation.Here, we explore a case-study of entrenched conflict where uncertainty exists over the impacts of multiple management actions: namely, moorlands managed for the shooting of red grouse (willow ptarmigan) Lagopus lagopus in the United Kingdom (UK). Debate over how UK moorlands should be managed is increasingly polarized. We evaluate, for the first time at a regional scale, the relative impacts of two major moorland management practices-predator control and heather burning-on nontarget bird species of conservation concern.Birds were surveyed on 18 estates across Northern England and Southeast Scotland. Sites ranged from intensively managed grouse moors to moorland sites with no management for grouse shooting. We hypothesised that both targeted predator control and burning regimes would enhance ground-nesting wader numbers and, as a consequence of this, and of increased grouse numbers, nontarget avian predators should also be more abundant on heavily managed sites.There were positive associations between predator control and the abundance of the three most widespread species of ground-nesting wader: strong effects for European golden plover Pluvialis apricaria and Eurasian curlew Numenius arquata and, less strongly, for common snipe Gallinago gallinago. These effects saturated at low levels of predator control. Evidence for effects of burning was much weaker. We found no evidence of enhanced numbers of nontarget predators on heavily managed sites.

Chemistry of nitric oxide and related species.

Nitric oxide (NO) has essential roles in a remarkable number of diverse biological processes. The reactivity of NO depends upon its physical properties, such as its small size, high diffusion rate, and lipophilicity (resulting in its accumulation in hydrophobic regions), and also on its facile but selective chemical reactivity toward a variety of cellular targets. NO also undergoes reactions with oxygen, superoxide ions, and reducing agents to give products that themselves show distinctive reactivity toward particular targets, sometimes with the manifestation of toxic effects, such as nitrosative stress. These include nitroxyl (HNO), the oxides NO2/N2O4, and N2O3, peroxynitrite, and S-nitrosothiols (RSNO). HNO is attracting considerable attention due to its pharmacological properties, which appear to be distinct from those of NO, and that may be significant in the treatment of heart failure.

The preparation and purification of NO gas and the use of NO releasers: the application of NO donors and other agents of nitrosative stress in biological systems.

Cylinders and lecture bottles are often the source of nitric oxide (NO) in studies of the biological chemistry of this remarkable molecule. The NO from both sources will probably contain NO2 (and N2O) formed by disproportionation of NO. The NO2 must be removed by passing the NO through a thoroughly deoxygenated sequence of traps containing sodium hydroxide solution and then water. The presence of NO2 in aqueous solutions of NO may be determined readily using 2,2' azino(3-ethylbenzothiazoline-6-sulfonic acid). NO2 oxidizes this compound to a long-lived radical anion, the concentration of which may be determined spectrophotometrically. The formation of NO by the decomposition of nitrous acid (via its disproportionation to nitrate and NO) and by the use of commercially available NO-releasing compounds with defined half-lives are also discussed. Other reactions that lead indirectly to the formation of NO are noted. In all cases, care must be taken to exclude oxygen to minimize as much as possible the formation of NO2 (and, consequently, the nitrosating agent N2O3). The uses of these methods for generating NO and the reactivity of related compounds are illustrated with examples of studies of nitrosative stress.

The Genetic Architecture Underlying the Evolution of a Rare Piscivorous Life History Form in Brown Trout after Secondary Contact and Strong Introgression.

Identifying the genetic basis underlying phenotypic divergence and reproductive isolation is a longstanding problem in evolutionary biology. Genetic signals of adaptation and reproductive isolation are often confounded by a wide range of factors, such as variation in demographic history or genomic features. Brown trout (Salmo trutta) in the Loch Maree catchment, Scotland, exhibit reproductively isolated divergent life history morphs, including a rare piscivorous (ferox) life history form displaying larger body size, greater longevity and delayed maturation compared to sympatric benthivorous brown trout. Using a dataset of 16,066 SNPs, we analyzed the evolutionary history and genetic architecture underlying this divergence. We found that ferox trout and benthivorous brown trout most likely evolved after recent secondary contact of two distinct glacial lineages, and identified 33 genomic outlier windows across the genome, of which several have most likely formed through selection. We further identified twelve candidate genes and biological pathways related to growth, development and immune response potentially underpinning the observed phenotypic differences. The identification of clear genomic signals divergent between life history phenotypes and potentially linked to reproductive isolation, through size assortative mating, as well as the identification of the underlying demographic history, highlights the power of genomic studies of young species pairs for understanding the factors shaping genetic differentiation.

CowPI: A Rumen Microbiome Focussed Version of the PICRUSt Functional Inference Software.

Metataxonomic 16S rDNA based studies are a commonplace and useful tool in the research of the microbiome, but they do not provide the full investigative power of metagenomics and metatranscriptomics for revealing the functional potential of microbial communities. However, the use of metagenomic and metatranscriptomic technologies is hindered by high costs and skills barrier necessary to generate and interpret the data. To address this, a tool for Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was developed for inferring the functional potential of an observed microbiome profile, based on 16S data. This allows functional inferences to be made from metataxonomic 16S rDNA studies with little extra work or cost, but its accuracy relies on the availability of completely sequenced genomes of representative organisms from the community being investigated. The rumen microbiome is an example of a community traditionally underrepresented in genome and sequence databases, but recent efforts by projects such as the Global Rumen Census and Hungate 1000 have resulted in a wide sampling of 16S rDNA profiles and almost 500 fully sequenced microbial genomes from this environment. Using this information, we have developed "CowPI," a focused version of the PICRUSt tool provided for use by the wider scientific community in the study of the rumen microbiome. We evaluated the accuracy of CowPI and PICRUSt using two 16S datasets from the rumen microbiome: one generated from rDNA and the other from rRNA where corresponding metagenomic and metatranscriptomic data was also available. We show that the functional profiles predicted by CowPI better match estimates for both the meta-genomic and transcriptomic datasets than PICRUSt, and capture the higher degree of genetic variation and larger pangenomes of rumen organisms. Nonetheless, whilst being closer in terms of predictive power for the rumen microbiome, there were differences when compared to both the metagenomic and metatranscriptome data and so we recommend, where possible, functional inferences from 16S data should not replace metagenomic and metatranscriptomic approaches. The tool can be accessed at http://www.cowpi.org and is provided to the wider scientific community for use in the study of the rumen microbiome.

Accuracy of two optical chlorophyll meters in predicting chemical composition and in vitro ruminal organic matter degradability of Brachiaria hybrid, Megathyrsus maximus, and Paspalum atratum.

The objective of this study was to determine the accuracy and reliability of 2 optical chlorophyll meters: FieldScout CM 1,000 NDVI and Yara N-Tester, in predicting neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL), acid detergent insoluble nitrogen (ADIN) and in vitro ruminal organic matter degradability (IVOMD) of 3 tropical grasses. Optical chlorophyll measurements were taken at 3 stages (4, 8 and 12 weeks) of regrowth in Brachiaria hybrid, and Megathyrsus maximus and at 6 and 12 weeks of regrowth in Paspalum atratum (cv. Ubon). Optical chlorophyll measurements showed the highest correlation (r = 0.57 to 0.85) with NDF concentration. The FieldScout CM 1,000 NDVI was better than the Yara N-Tester in predicting NDF (R2 = 0.70) and ADF (R2 = 0.79) concentrations in Brachiaria hybrid and NDF (R2 = 0.79) in M. maximus. Similarly, FieldScout CM 1,000 NDVI produced better estimates of 24 h IVOMD (IVOMD24h) in Brachiaria hybrid (R2 = 0.81) and IVOMD48h in Brachiaria hybrid (R2 = 0.65) and M. maximus (R2 = 0.75). However, these prediction models had relatively low concordance correlation coefficients, i.e., CCC >0.90, but random errors were the main source of bias. It was, therefore, concluded that both optical chlorophyll meters were poor and unreliable predictors of ADIN and ADL concentrations. Overall, the FieldScout CM 1,000 NDVI shows potential to produce useful estimates of IVOMD24h and ADF in Brachiaria hybrid and IVOMD48h and NDF concentrations in M. maximus.

Time-calibrated molecular phylogeny of pteropods.

Pteropods are a widespread group of holoplanktonic gastropod molluscs and are uniquely suitable for study of long-term evolutionary processes in the open ocean because they are the only living metazoan plankton with a good fossil record. Pteropods have been proposed as bioindicators to monitor the impacts of ocean acidification and in consequence have attracted considerable research interest, however, a robust evolutionary framework for the group is still lacking. Here we reconstruct their phylogenetic relationships and examine the evolutionary history of pteropods based on combined analyses of Cytochrome Oxidase I, 28S, and 18S ribosomal rRNA sequences and a molecular clock calibrated using fossils and the estimated timing of the formation of the Isthmus of Panama. Euthecosomes with uncoiled shells were monophyletic with Creseis as the earliest diverging lineage, estimated at 41-38 million years ago (mya). The coiled euthecosomes (Limacina, Heliconoides, Thielea) were not monophyletic contrary to the accepted morphology-based taxonomy; however, due to their high rate heterogeneity no firm conclusions can be drawn. We found strong support for monophyly of most euthecosome genera, but Clio appeared as a polyphyletic group, and Diacavolinia grouped within Cavolinia, making the latter genus paraphyletic. The highest evolutionary rates were observed in Heliconoides inflatus and Limacina bulimoides for both 28S and 18S partitions. Using a fossil-calibrated phylogeny that sets the first occurrence of coiled euthecosomes at 79-66 mya, we estimate that uncoiled euthecosomes evolved 51-42 mya and that most extant uncoiled genera originated 40-15 mya. These findings are congruent with a molecular clock analysis using the Isthmus of Panama formation as an independent calibration. Although not all phylogenetic relationships could be resolved based on three molecular markers, this study provides a useful resource to study pteropod diversity and provides general insight into the processes that generate and maintain their diversity in the open ocean.

Inhibitory effects of sulfamic acid on three thiosulfate-oxidizing chemolithotrophs.

Sulfamate is an analogue of thiosulfate, and the sodium and potassium salts of sulfamic acid inhibited the chemolithoautotrophic growth on thiosulfate of Acidithiobacillus ferrooxidans and Halothiobacillus neapolitanus. The chemo-organotrophic growth of Paracoccus versutus on sucrose was similarly inhibited by sulfamate. Thiosulfate oxidation by suspensions of H. neapolitanus was, however, unaffected by sulfamate, showing that sulfamate did not directly affect thiosulfate uptake, activation or oxidation. Inhibition of P. versutus was not relieved by cysteine and methionine, indicating that sulfate uptake and sulfur amino acid biosynthesis were not directly affected by sulfamate. Sulfamate was not degraded by any of the bacteria, and so could not serve as an alternative to thiosulfate as an energy-yielding substrate. Sulfamate is also an analogue of ammonia and might act like hydrazine by inhibiting ammonium uptake or an essential enzyme activity.

Recombinant human activated protein C.

This review will summarise the relevant pathophysiology of sepsis, the rationale for treatment with recombinant human activated protein C and the evidence for and against its use, and will provide evidence-based recommendations for its administration.

What limits the morphological disparity of clades?

The morphological disparity of species within major clades shows a variety of trajectory patterns through evolutionary time. However, there is a significant tendency for groups to reach their maximum disparity relatively early in their histories, even while their species richness or diversity is comparatively low. This pattern of early high-disparity suggests that there are internal constraints (e.g. developmental pleiotropy) or external restrictions (e.g. ecological competition) upon the variety of morphologies that can subsequently evolve. It has also been demonstrated that the rate of evolution of new character states decreases in most clades through time (character saturation), as does the rate of origination of novel bodyplans and higher taxa. Here, we tested whether there was a simple relationship between the level or rate of character state exhaustion and the shape of a clade's disparity profile: specifically, its centre of gravity (CG). In a sample of 93 extinct major clades, most showed some degree of exhaustion, but all continued to evolve new states up until their extinction. Projection of states/steps curves suggested that clades realized an average of 60% of their inferred maximum numbers of states. Despite a weak but significant correlation between overall levels of homoplasy and the CG of clade disparity profiles, there were no significant relationships between any of our indices of exhaustion curve shape and the clade disparity CG. Clades showing early high-disparity were no more likely to have early character saturation than those with maximum disparity late in their evolution.

Kinetics of the reactions of nitrogen dioxide with glutathione, cysteine, and uric acid at physiological pH.

Nitrogen dioxide (NO(2)(*)) is a key biological oxidant. It can be derived from peroxynitrite via the interaction of nitric oxide with superoxide, from nitrite with peroxidases, or from autoxidation of nitric oxide. In this study, submicromolar concentrations of NO(2)(*) were generated in < 1 micros using pulse radiolysis, and the kinetics of scavenging NO(2)(*) by glutathione, cysteine, or uric acid were monitored by spectrophotometry. The formation of the urate radical was observed directly, while the production of the oxidizing radical obtained on reaction of NO(2)(*) with the thiols (the thiyl radical) was monitored via oxidation of 2,2'-azino-bis-(3-ethylthiazoline-6-sulfonic acid). At pH 7.4, rate constants for reaction of NO(2)(*) with glutathione, cysteine, and urate were estimated as approximately 2 x 10(7), 5 x 10(7), and 2 x 10(7) M(-1) s(-1), respectively. The variation of these rate constants with pH indicated that thiolate reacted much faster than undissociated thiol. The dissociation of urate also accelerated reaction with NO(2)(*) at pH > 8. The thiyl radical from GSH reacted with urate with a rate constant of approximately 3 x 10(7) M(-1) s(-1). The implications of these values are: (i) the lifetime of NO(2)(*) in cytosol is < 10 micros; (ii) thiols are the dominant 'sink' for NO(2)(*) in cells/tissue, whereas urate is also a major scavenger in plasma; (iii) the diffusion distance of NO(2)(*) is approximately 0.2 microm in the cytoplasm and < 0.8 microm in plasma; (iv) urate protects GSH against depletion on oxidative challenge from NO(2)(*); and (v) reactions between NO(2)(*) and thiols/urate severely limit the likelihood of reaction of NO(2)(*) with NO* to form N(2)O(3) in the cytoplasm.

Interaction of bilirubin and biliverdin with reactive nitrogen species.

Bilirubin (BR) and biliverdin (BV), two metabolites produced during haem degradation by haem oxygenase, possess strong antioxidant activities toward peroxyl radical, hydroxyl radical and hydrogen peroxide. Considering the importance attributed to nitric oxide (NO) and its congeners in the control of physiological and pathophysiological processes, we examined the interaction of BR and BV with NO and NO-related species in vitro. Exposure of BR and BV to agents that release NO or nitroxyl resulted in a concentration- and time-dependent loss of BR and BV, as assessed by high performance liquid chromatography. Peroxynitrite, a strong oxidant derived from the reaction of NO with superoxide anion, also showed high reactivity toward BR and BV. The extent of BR and BV consumption largely depended on the NO species being analysed and on the half-lives of the pharmacological compounds considered. Of major importance, BR and BV decomposition occurred also in the presence of pure NO under anaerobic conditions, confirming the ability of bile pigments to scavenge the gaseous free radical. Increasing concentrations of thiols prevented BR consumption by nitroxyl, indicating that bile pigments and thiol groups can compete and/or synergise the cellular defence against NO-related species. In view of the high inducibility of haem oxygenase-1 by NO-releasing agents in different cell types, the present findings highlight novel anti-nitrosative characteristics of BR and BV suggesting a potential function for bile pigments against the damaging effects of uncontrolled NO production.