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Bacterial microcompartments are prokaryotic organelles comprising encapsulated enzymes within a thin protein shell. They facilitate metabolic processing including propanediol, choline, glycerol, and ethanolamine utilization, and they accelerate carbon fixation in cyanobacteria. Enzymes targeted to the inside of the microcompartment frequently possess a cargo-encapsulation peptide, but the site to which the peptide binds is unclear. We provide evidence that the encapsulation peptides bind to the hydrophobic groove formed between tessellating subunits of the shell proteins. In silico docking studies provide a compelling model of peptide binding to this prominent hydrophobic groove. This result is consistent with the now widely accepted view that the convex side of the shell oligomers faces the lumen of the microcompartment. Binding of the encapsulation peptide to the groove between tessellating shell protein tiles explains why it has been difficult to define the peptide binding site using other methods, provides a mechanism by which encapsulation-peptide bearing enzymes can promote shell assembly, and explains how the presence of cargo affects the size and shape of the bacterial microcompartment. This knowledge may be exploited in engineering microcompartments or in disease prevention by hampering cargo-encapsulation.

Vitamin B12 is an essential nutrient that is not made by plants; consequently, unfortified plant-based foods are not a reliable supply. Recent estimates suggest high rates of vitamin B12 deficiency among the vegetarian and vegan populations, particularly in pregnant women or women of child-bearing age who, for ethical and health reasons, are shifting towards higher consumption of plant-based foods in ever-increasing numbers. Vitamin B12 plays crucial metabolic roles across the life-course and in particular during pregnancy and in early development (first 1000 days of life). Evidence now implicates vitamin B12 deficiency with increased risk to a range of neuro, vascular, immune, and inflammatory disorders. However, the current UK recommended nutrient intake for vitamin B12 does not adequately consider the vitamin B12 deficit for those choosing a plant-based diet, including vegetarianism and in particular veganism, representing a hidden hunger. We provide a cautionary note on the importance of preventing vitamin B12 deficits for those individuals choosing a plant-based diet and the health professionals advising them.

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling. Despite their phototrophic lifestyle, over half of algal species depend for growth on acquiring an external supply of the corrinoid vitamin B12 (cobalamin), a micronutrient produced only by a subset of prokaryotic organisms. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about its uptake in algae. Here, we demonstrate the essential role of a protein, cobalamin acquisition protein 1 (CBA1), in B12 uptake in Phaeodactylum tricornutum using CRISPR-Cas9 to generate targeted knockouts and in Chlamydomonas reinhardtii by insertional mutagenesis. In both cases, CBA1 knockout lines could not take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wild-type CBA1 gene restored B12 uptake, and regulation of CBA1 expression via a riboswitch element enabled control of the phenotype. When visualised by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 showed association with membranes. Bioinformatics analysis found that CBA1-like sequences are present in all major eukaryotic phyla. In algal taxa, the majority that encoded CBA1 also had genes for B12-dependent enzymes, suggesting CBA1 plays a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities.

The natural vitamin B12‐derivatives are intriguing complexes of cobalt that entrap the metal within the strikingly skewed and ring‐contracted corrin ligand. Here, we describe the synthesis of the Rh(III)‐corrin acetylrhodibalamin (AcRhbl) from biotechnologically produced metal‐free hydrogenobyric acid and analyze the effect of the replacement of the cobalt‐center of the organometallic vitamin B12‐derivative acetylcobalamin (AcCbl) with its group‐IX homologue rhodium, to give AcRhbl. The structures of AcCbl and AcRhbl were thoroughly analyzed in aqueous solution, in crystals and by in silico methods, in order to gain detailed insights into the structural adaptations to the two homologous metals. Indeed, the common, nucleotide‐appended corrin‐ligand in these two metal corrins features extensive structural similarity. Thus, the rhodium‐corrin AcRhbl joins the small group of B12‐mimics classified as ‘antivitamins B12’, isostructural metal analogues of the natural cobalt‐corrins that hold significant potential in biological and biomedical applications as selective inhibitors of key cellular processes.

The natural vitamin B12‐derivatives are intriguing complexes of cobalt that entrap the metal within the strikingly skewed and ring‐contracted corrin ligand. Here, we describe the synthesis of the Rh(III)‐corrin acetylrhodibalamin (AcRhbl) from biotechnologically produced metal‐free hydrogenobyric acid and analyze the effect of the replacement of the cobalt‐center of the organometallic vitamin B12‐derivative acetylcobalamin (AcCbl) with its group‐IX homologue rhodium, to give AcRhbl. The structures of AcCbl and AcRhbl were thoroughly analyzed in aqueous solution, in crystals and by in silico methods, in order to gain detailed insights into the structural adaptations to the two homologous metals. Indeed, the common, nucleotide‐appended corrin‐ligand in these two metal corrins features extensive structural similarity. Thus, the rhodium‐corrin AcRhbl joins the small group of B12‐mimics classified as ‘antivitamins B12’, isostructural metal analogues of the natural cobalt‐corrins that hold significant potential in biological and biomedical applications as selective inhibitors of key cellular processes.

We review changes in the status of butterflies in Europe, focusing on long-running population data available for the United Kingdom, the Netherlands, and Belgium, based on standardized monitoring transects. In the United Kingdom, 8% of resident species have become extinct, and since 1976 overall numbers declined by around 50%. In the Netherlands, 20% of species have become extinct, and since 1990 overall numbers in the country declined by 50%. Distribution trends showed that butterfly distributions began decreasing long ago, and between 1890 and 1940, distributions declined by 80%. In Flanders (Belgium), 20 butterflies have become extinct (29%), and between 1992 and 2007 overall numbers declined by around 30%. A European Grassland Butterfly Indicator from 16 European countries shows there has been a 39% decline of grassland butterflies since 1990. The 2010 Red List of European butterflies listed 38 of the 482 European species (8%) as threatened and 44 species (10%) as near threatened (note that 47 species were not assessed). A country level analysis indicates that the average Red List rating is highest in central and mid-Western Europe and lowest in the far north of Europe and around the Mediterranean. The causes of the decline of butterflies are thought to be similar in most countries, mainly habitat loss and degradation and chemical pollution. Climate change is allowing many species to spread northward while bringing new threats to susceptible species. We describe examples of possible conservation solutions and a summary of policy changes needed to conserve butterflies and other insects.

The situated nature of professional and other workplace-based communication continues to receive attention in intercultural communication research (see, e.g., Cheng and Kong 2009; Ladegaard and Jenks 2017; Schnurr and Zayts 2017). Inevitably, these studies provide accounts of the intercultural nature of such communication which go beyond ‘macro categories’ and adopt a more emic perspective (Cheng and Kong 2009: 8). Studies which highlight and seek to explain the complexities of intercultural workplace communication are the focus of this chapter. The authors provide an overview of the types of studies conducted to date, paying attention to where, and how, data were collected and the extent to which participants were involved in the analysis of the data as advocated by Sarangi (2002: 99). Schnurr and Zayts (2017: 148) describe the complexities which need to be understood if we are to better understand language and culture in the workplace. These are the dynamic norms, values, and behaviours of professionals; the nature of the professional contexts and workplaces in which intercultural communication takes place; and the specific context in which each interaction takes place. These aspects are foregrounded when the studies are reviewed and conclusions and implications for future research are discussed.

The situated nature of professional and other workplace-based communication continues to receive attention in intercultural communication research (see, e.g., Cheng and Kong 2009; Ladegaard and Jenks 2017; Schnurr and Zayts 2017). Inevitably, these studies provide accounts of the intercultural nature of such communication which go beyond ‘macro categories’ and adopt a more emic perspective (Cheng and Kong 2009: 8). Studies which highlight and seek to explain the complexities of intercultural workplace communication are the focus of this chapter. The authors provide an overview of the types of studies conducted to date, paying attention to where, and how, data were collected and the extent to which participants were involved in the analysis of the data as advocated by Sarangi (2002: 99). Schnurr and Zayts (2017: 148) describe the complexities which need to be understood if we are to better understand language and culture in the workplace. These are the dynamic norms, values, and behaviours of professionals; the nature of the professional contexts and workplaces in which intercultural communication takes place; and the specific context in which each interaction takes place. These aspects are foregrounded when the studies are reviewed and conclusions and implications for future research are discussed.

Red Lists are very valuable tools in nature conservation at global, continental and (sub-) national scales. In an attempt to prioritise conservation actions for European butterflies, we compiled a database with species lists and Red Lists of all European countries, including the Macaronesian archipelagos (Azores, Madeira and Canary Islands). In total, we compiled national species lists for 42 countries and national Red Lists for 34 of these. The most species-rich countries in Europe are Italy, Russia and France with more than 250 species each. Endemic species are mainly found on the Macaronesian archipelagos and on the Mediterranean islands. By attributing numerical values proportionate to the threat statuses in the different national Red List categories, we calculated a mean Red List value for every country (cRLV) and a weighted Red List value for every species (wsRLV) using the square root of the country’s area as a weighting factor. Countries with the highest cRLV were industrialised (NW) European countries such as the Netherlands, Belgium, the Czech Republic and Denmark, whereas large Mediterranean countries such as Spain and Italy had the lowest cRLV. Species for which a Red List assessment was available in at least two European countries and with a relatively high wsRLV (≥ 50) are Colias myrmidone, Pseudochazara orestes, Tomares nogelii, Colias chrysotheme and Coenonympha oedippus. We compared these wsRLVs with the species statuses on the European Red List to identify possible mismatches. We discuss how this complementary method can help to prioritise butterfly conservation on the continental and/or the (sub-)national scale.

Red Lists are very valuable tools in nature conservation at global, continental and (sub-) national scales. In an attempt to prioritise conservation actions for European butterflies, we compiled a database with species lists and Red Lists of all European countries, including the Macaronesian archipelagos (Azores, Madeira and Canary Islands). In total, we compiled national species lists for 42 countries and national Red Lists for 34 of these. The most species-rich countries in Europe are Italy, Russia and France with more than 250 species each. Endemic species are mainly found on the Macaronesian archipelagos and on the Mediterranean islands. By attributing numerical values proportionate to the threat statuses in the different national Red List categories, we calculated a mean Red List value for every country (cRLV) and a weighted Red List value for every species (wsRLV) using the square root of the country's area as a weighting factor. Countries with the highest cRLV were industrialised (NW) European countries such as the Netherlands, Belgium, the Czech Republic and Denmark, whereas large Mediterranean countries such as Spain and Italy had the lowest cRLV. Species for which a Red List assessment was available in at least two European countries and with a relatively high wsRLV (50) are Colias myrmidone, Pseudochazara orestes, Tomares nogelii, Colias chrysotheme and Coenonympha oedippus. We compared these wsRLVs with the species statuses on the European Red List to identify possible mismatches. We discuss how this complementary method can help to prioritise butterfly conservation on the continental and/or the (sub-)national scale.

Bacterial microcompartments (BMCs) represent proteinaceous macromolecular nano-bioreactors that are found in a broad range of bacteria, and which are associated with either anabolic or catabolic processes. They consist of a semipermeable outer shell that packages a central metabolic enzyme or pathway, providing both enhanced flux and protection against toxic intermediates. Recombinant production of BMCs has led to their repurposing with the incorporation of altogether new pathways. Deconstruction of BMCs into their component parts has shown that some individual shell proteins self-associate into filaments that can be further modified into a cytoplasmic scaffold to which enzymes/proteins can be targeted. BMCs therefore represent a modular system that is highly suited for the engineering of biological systems for useful purposes.

Bacterial microcompartments, BMCs, are proteinaceous organelles that encase a specific metabolic pathway within a semi-permeable protein shell. Short encapsulation peptides can direct cargo proteins to the lumen of the compartments. However, the fusion of such peptides to non-native proteins does not guarantee encapsulation and often causes aggregation. Here, we report an approach for targeting recombinant proteins to BMCs that utilizes specific de novo coiled-coil protein–protein interactions. Attachment of one coiled-coil module to PduA (a component of the BMC shell) allows targeting of a fluorescent protein fused to a cognate coiled-coil partner. This interaction takes place on the outer surface of the BMC. The redesign of PduA to generate an N-terminus on the luminal side of the BMC results in intact compartments to which proteins can still be targeted via the designed coiled-coil system. This study provides a strategy to display proteins on the surface or within the lumen of the BMCs.

The biosynthesis of B12, involving up to 30 different enzyme-mediated steps, only occurs in bacteria. Thus, most eukaryotes require an external source of B12, and yet the vitamin appears to have only two functions in eukaryotes: as a cofactor for the enzymes methionine synthase and methylmalonylCoA mutase. These two functions are crucial for normal health in humans, and in particular, the formation of methionine is essential for providing methyl groups for over 100 methylation processes. Interference with the methionine synthase reaction not only depletes the body of methyl groups but also leads to the accumulation of homocysteine, a risk factor for many diseases. The syndrome pernicious anemia, characterized by lack of intrinsic factor, leads to a severe, sometimes fatal form of B12 deficiency. However, there is no sharp cutoff for B12 deficiency; rather, there is a continuous inverse relationship between serum B12 and a variety of undesirable outcomes, including neural tube defects, stroke, and dementia. The brain is particularly vulnerable; in children, inadequate B12 stunts brain and intellectual development. Suboptimal B12 status (serum B12 < 300 pmol/L) is very common, occurring in 30%–60% of the population, in particular in pregnant women and in less-developed countries. Thus, many tens of millions of people in the world may suffer harm from having a poor B12 status. Public health steps are urgently needed to correct this inadequacy.

This report presents the fifth version of the European Grassland Butterfly Indicator, one of the EU biodiversity indicators of the European Environment Agency.

This report presents the fifth version of the European Grassland Butterfly Indicator, one of the EU biodiversity indicators of the European Environment Agency.

Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the solution structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase associated with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The solution structure reveals the peptide to have a well-defined helical conformation along its whole length. Saturation transfer difference and transferred NOE NMR has highlighted the observed interaction surface on the peptide with its main interacting shell protein, PduK. By tagging both a pyruvate decarboxylase and an alcohol dehydrogenase with targeting peptides, it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Not only are the purified, redesigned BMCs able to transform pyruvate into ethanol efficiently, but the strains containing the modified BMCs produce elevated levels of alcohol.

Adenosylcobalamin, the coenzyme form of vitamin B12, is one Nature's most complex coenzyme whose de novo biogenesis proceeds along either an anaerobic or aerobic metabolic pathway. The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion. Herein, we reveal how Brucella melitensis CobR binds its coenzyme FAD (flavin dinucleotide) and we also show that the enzyme can bind a corrin substrate consistent with its role in reduction of the cobalt of the corrin ring. Stopped-flow kinetics and EPR reveal a mechanistic asymmetry in CobR dimer that provides a potential link between the two electron reduction by NADH to the single electron reduction of Co(II) to Co(I).

Recent investigations from modern environments demonstrate that many terminal moraines do not simply record a single glacial maximum, but instead reveal a complex oscillatory pattern of ice-marginal behaviour including polyphase retreat. Within this study, we examine the geomorphology, geology and internal structure of a terminal moraine complex – the ‘Cromer Ridge’ in north Norfolk to reconstruct patterns of ice-marginal behaviour. Previously, this landform was interpreted as the limit of a southern extension of the British Ice Sheet during a Middle Pleistocene glaciation. Evidence presented here reveals a more complicated pattern of ice-marginal behaviour with the ‘Cromer Ridge’ reinterpreted as a ‘complex’ comprising several ridge elements. We propose that the maximum ice extent lay further to the south, with the size and morphology of the largest ridge element (the ‘Cromer Ridge’ as previously defined) a facet of thrust-stacking at an ice-marginal still-stand. We recognise multiple oscillations of the ice-front recorded against a twelve-stage model for the decay of the southern margins of a fast-flowing lobe of North Sea ice. Changes in ice-marginal dynamics are identified by the superimposition and lateral and vertical evolution of glacitectonic styles. Differences between these various states, and switches between ‘shallow’ and ‘deep’ thin-skinned glacitectonics, are strongly influenced by sub-marginal and proglacial water availability. Examination of the evidence for the morphostratigraphic proposals for the glacitectonic assemblage, within the context of the above interpretation, suggests that many of the ‘glacigenic landforms’ are erosional and a MIS 12 age of formation is favoured although several anomalies remain to be explained.

Recent investigations from modern environments demonstrate that many terminal moraines do not simply record a single glacial maximum, but instead reveal a complex oscillatory pattern of ice-marginal behaviour including polyphase retreat. Within this study, we examine the geomorphology, geology and internal structure of a terminal moraine complex – the ‘Cromer Ridge’ in north Norfolk to reconstruct patterns of ice-marginal behaviour. Previously, this landform was interpreted as the limit of a southern extension of the British Ice Sheet during a Middle Pleistocene glaciation. Evidence presented here reveals a more complicated pattern of ice-marginal behaviour with the ‘Cromer Ridge’ reinterpreted as a ‘complex’ comprising several ridge elements. We propose that the maximum ice extent lay further to the south, with the size and morphology of the largest ridge element (the ‘Cromer Ridge’ as previously defined) a facet of thrust-stacking at an ice-marginal still-stand. We recognise multiple oscillations of the ice-front recorded against a twelve-stage model for the decay of the southern margins of a fast-flowing lobe of North Sea ice. Changes in ice-marginal dynamics are identified by the superimposition and lateral and vertical evolution of glacitectonic styles. Differences between these various states, and switches between ‘shallow’ and ‘deep’ thin-skinned glacitectonics, are strongly influenced by sub-marginal and proglacial water availability. Examination of the evidence for the morphostratigraphic proposals for the glacitectonic assemblage, within the context of the above interpretation, suggests that many of the ‘glacigenic landforms’ are erosional and a MIS 12 age of formation is favoured although several anomalies remain to be explained.

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.

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.

Twenty-nine butterfly species are listed on the Annexes of the Habitats Directive. To assist everyone who wants or needs to take action for one of these species, we compiled an overview of the habitat requirements and ecology of each species, as well as information on their conservation status in Europe. This was taken from the recent Red List and their main biogeographical regions (taken from the first reporting on Article 17 of the Directive). Most important are the Dos and Don`ts, which summarize in a few bullet points what to do and what to avoid in order to protect and conserve these butterflies and their habitats.

Sulfate-reducing microorganisms are a diverse group of bacteria and archaea that occupy important environmental niches and have potential for significant biotechnological impact. Desulfovibrio, the most studied genus among the sulfate-reducing microorganisms, contains proteins with a wide variety of tetrapyrrole-derived cofactors, including some unique derivatives such as uroporphyrin I and coproporphyrin III. Herein, we review tetrapyrrole metabolism in Desulfovibrio spp., including the production of sirohaem and cobalamin, and compare and contrast the biochemical properties of the enzymes involved in these biosynthetic pathways. Furthermore, we describe a novel pathway used by Desulfovibrio to synthesize haem b, which provides a previously unrecognized link between haem, sirohaem, and haem d(1). Finally, the organization and regulation of genes involved in the tetrapyrrole biosynthetic pathway is discussed.

1. Over the last century butterflies have undergone substantial changes in abundance and range in Great Britain and monitoring has improved markedly. These changes, together with a major revision of International Union for Conservation of Nature (IUCN) criteria, render previous Red List assessments outdated. 2. A new Red List assessment of all 62 resident and regularly breeding butterfly species in Britain was undertaken. The current IUCN criteria were applied for the first time to British butterflies using data from the Butterflies for the New Millennium recording scheme and the UK Butterfly Monitoring Scheme. 3. The state of knowledge and nature of the data available from these two schemes enabled assessment to be based upon two quantitative IUCN criteria: A2 (rate of population decline) and B2 (area of occupancy). 4. Twenty-three species (37% of the total) qualified as Regionally Extinct (RE) or threatened: 4 species were listed as RE and 19 as threatened (two Critically Endangered, eight Endangered and nine Vulnerable). A further 11 species were classified as Near Threatened. The remaining 28 species (45% of the total) were classified as Least Concern. 5. In comparison with previous assessments, we show that the number of species classified as threatened has increased. This is likely to be due to a combination of refined assessment criteria, improved data, and a real increase in the extinction threat as a result of human impacts. 6. The results confirm butterflies as a highly threatened group of insects in Great Britain and the Red List provides an important foundation to define conservation priorities.

1. Over the last century butterflies have undergone substantial changes in abundance and range in Great Britain and monitoring has improved markedly. These changes, together with a major revision of International Union for Conservation of Nature (IUCN) criteria, render previous Red List assessments outdated. 2. A new Red List assessment of all 62 resident and regularly breeding butterfly species in Britain was undertaken. The current IUCN criteria were applied for the first time to British butterflies using data from the Butterflies for the New Millennium recording scheme and the UK Butterfly Monitoring Scheme. 3. The state of knowledge and nature of the data available from these two schemes enabled assessment to be based upon two quantitative IUCN criteria: A2 (rate of population decline) and B2 (area of occupancy). 4. Twenty-three species (37% of the total) qualified as Regionally Extinct (RE) or threatened: 4 species were listed as RE and 19 as threatened (two Critically Endangered, eight Endangered and nine Vulnerable). A further 11 species were classified as Near Threatened. The remaining 28 species (45% of the total) were classified as Least Concern. 5. In comparison with previous assessments, we show that the number of species classified as threatened has increased. This is likely to be due to a combination of refined assessment criteria, improved data, and a real increase in the extinction threat as a result of human impacts. 6. The results confirm butterflies as a highly threatened group of insects in Great Britain and the Red List provides an important foundation to define conservation priorities.

Report no VS2010.037

Cobalamins belong to the tetrapyrrole family of prosthetic groups. The presence of a metal ion is a key feature of these compounds. In the oxygen-dependent (aerobic) cobalamin biosynthetic pathway, cobalt is inserted into a ring-contracted tetrapyrrole called hydrogenobyrinic acid a,c-diamide (HBAD) by a cobaltochelatase that is constituted by three subunits, CobN, CobS and COT, with molecular masses of 137, 37 and 71 kDa, respectively. Based on the similarities with magnesium chelatase, cobaltochelatase has been suggested to belong to the AAA(+) superfamily of proteins. In this paper we present the cloning of the Brucella melitensis cobN, cobS and cobT, the purification of the encoded protein products, and a single-particle reconstruction of the macromolecular assembly formed between CobS and COT from negatively stained electron microscopy images of the complex. The results show for the first time that subunits CobS and COT form a chaperone-like complex, characteristic for the AAA(+) class of proteins. The molecules are arranged in a two-tiered ring structure with the six subunits in each ring organized as a trimer of dimers. The similarity between this structure and that of magnesium chelatase, as well as analysis of the amino acid sequences confirms the suggested evolutionary relationship between the two enzymes., QC 20101213

La investigació en fraseologia i intertextualitat cobra cada vegada major interès. Aquest article analitza les conseqüències de combinar ambdós fenòmens en l'aprenentatge i investigació de l'anglès per a la comunicació professional. A partir de corpus recopilats pel Research Centre for Professional Communication in English, es recorre al concepte recentment desenvolupat de congramming (Cheng, Greaves i Warren, 2006; Cheng, Greaves, Sinclair i Warren, en premsa) per a estudiar de quina manera s'anota la intertextualitat en un corpus de fluxos discursius, que són una sèrie de discursos interconnectats. Els fluxos discursius del present estudi es van obtenir d'un professional durant una setmana. Els concgrams arrepleguen tota la variació constituent (AB, A*B, A**B, etc.) i la variació posicional (és a dir, AB, BA) de les paraules coocurrents en un text o corpus. Amb l'ús de concgrams extrets del corpus del flux discursiu, es presenten exemples de fraseologia i es discuteix la seua funció potencial en l?anàlisi dels casos d?intertextualitat en els textos., There is increasing interest in researching phraseology and intertextuality, but they are not usually studied together. This paper explores the implications of combining the two in the learning and teaching of English for professional communication. Using data compiled at the Hong Kong-based Research Centre for Professional Communication in English, in combination with the recently developed corpus linguistics methodology of ?congramming? (Cheng, Greaves and Warren, 2006; Cheng, Greaves, Sinclair and Warren, 2009), this study investigates how intertextuality can be signalled in a corpus of discourse flows. A discourse flow is a series of interconnected discourses and the flows in this study were collected from a professional over a period of one week. Concgramming is the process of fully automatically identifying concgrams in a text or corpus. Concgrams are co-occurrences of words (e.g. hard and work) irrespective of any constituent variation (work hard, work very hard, work so very hard, etc.) and positional variation (i.e. work hard, hard work, etc.) that might be present. Using concgrams extracted from the discourse flow corpus, examples of frequent phraseologies associated with the signalling of intertextuality are identified and their role in the realisation of intertextuality discussed., La investigación en fraseología e intertextualidad cobra cada vez mayor interés. Este artículo analiza las consecuencias de combinar ambos fenómenos en el aprendizaje e investigación del inglés para la comunicación profesional. A partir de corpus recopilados por el Research Centre for Professional Communication in English se recurre al concepto recientemente desarrollado de congramming (Cheng, Greaves y Warren, 2006; Cheng, Greaves, Sinclair y Warren, en prensa) para estudiar de qué modo se anota la intertextualidad en un corpus de flujos discursivos, que son una serie de discursos interconectados. Los flujos discursivos del presente estudio se obtuvieron de un profesional a lo largo de una semana. En cuanto a los concgrams, estos recogen toda la variación constituyente (AB, A*B, A**B, etc.) y la variación posicional (es decir, AB, BA) de las palabras coocurrentes en un texto o corpus. Utilizando concgrams extraídos del corpus de flujos discursivos, se identifican y discuten ejemplos de las fraseologías más frecuentes asociadas con la señalización de la intertextualidad.

La investigació en fraseologia i intertextualitat cobra cada vegada major interès. Aquest article analitza les conseqüències de combinar ambdós fenòmens en l'aprenentatge i investigació de l'anglès per a la comunicació professional. A partir de corpus recopilats pel Research Centre for Professional Communication in English, es recorre al concepte recentment desenvolupat de congramming (Cheng, Greaves i Warren, 2006; Cheng, Greaves, Sinclair i Warren, en premsa) per a estudiar de quina manera s'anota la intertextualitat en un corpus de fluxos discursius, que són una sèrie de discursos interconnectats. Els fluxos discursius del present estudi es van obtenir d'un professional durant una setmana. Els concgrams arrepleguen tota la variació constituent (AB, A*B, A**B, etc.) i la variació posicional (és a dir, AB, BA) de les paraules coocurrents en un text o corpus. Amb l'ús de concgrams extrets del corpus del flux discursiu, es presenten exemples de fraseologia i es discuteix la seua funció potencial en l?anàlisi dels casos d?intertextualitat en els textos., There is increasing interest in researching phraseology and intertextuality, but they are not usually studied together. This paper explores the implications of combining the two in the learning and teaching of English for professional communication. Using data compiled at the Hong Kong-based Research Centre for Professional Communication in English, in combination with the recently developed corpus linguistics methodology of ?congramming? (Cheng, Greaves and Warren, 2006; Cheng, Greaves, Sinclair and Warren, 2009), this study investigates how intertextuality can be signalled in a corpus of discourse flows. A discourse flow is a series of interconnected discourses and the flows in this study were collected from a professional over a period of one week. Concgramming is the process of fully automatically identifying concgrams in a text or corpus. Concgrams are co-occurrences of words (e.g. hard and work) irrespective of any constituent variation (work hard, work very hard, work so very hard, etc.) and positional variation (i.e. work hard, hard work, etc.) that might be present. Using concgrams extracted from the discourse flow corpus, examples of frequent phraseologies associated with the signalling of intertextuality are identified and their role in the realisation of intertextuality discussed., La investigación en fraseología e intertextualidad cobra cada vez mayor interés. Este artículo analiza las consecuencias de combinar ambos fenómenos en el aprendizaje e investigación del inglés para la comunicación profesional. A partir de corpus recopilados por el Research Centre for Professional Communication in English se recurre al concepto recientemente desarrollado de congramming (Cheng, Greaves y Warren, 2006; Cheng, Greaves, Sinclair y Warren, en prensa) para estudiar de qué modo se anota la intertextualidad en un corpus de flujos discursivos, que son una serie de discursos interconectados. Los flujos discursivos del presente estudio se obtuvieron de un profesional a lo largo de una semana. En cuanto a los concgrams, estos recogen toda la variación constituyente (AB, A*B, A**B, etc.) y la variación posicional (es decir, AB, BA) de las palabras coocurrentes en un texto o corpus. Utilizando concgrams extraídos del corpus de flujos discursivos, se identifican y discuten ejemplos de las fraseologías más frecuentes asociadas con la señalización de la intertextualidad.

Biotin synthase (BioB) catalyses the final step in the biosynthesis of biotin. Aerobically purified biotin synthase contains one [2Fe–2S]2+ cluster per monomer. However, active BioB contains in addition a [4Fe–4S]2+ cluster which can be formed either by reconstitution with iron and sulfide, or on reduction with sodium dithionite. Here, we use EPR spectroscopy to show that mutations in the conserved YNHNLD sequence of Escherichia coli BioB affect the formation and stability of the [4Fe–4S]1+ cluster on reduction with dithionite and report the observation of a new [2Fe–2S]1+ cluster. These results serve to illustrate the dynamic nature of iron–sulfur clusters in biotin synthase and the role played by the protein in cluster interconversion.

The Chalkhill Blue Polyommatus coridon is a widespread butterfly of lowland calcareous grassland in southern Britain and is considered a good indicator of habitat condition. Polyommatus coridon has been identified as a Species of Conservation Concern in the UK Biodiversity Action Plan due to a greater than 25% decline in range size since the 1950s, with losses due to the combined effects of habitat destruction, agricultural intensification and neglect. Analysis of annual butterfly monitoring data (transects) collected at 161 sites from 1981 to 2000 show a three-fold population recovery had occurred. The increases were at established sites, with no re-colonisations or range expansion detected. The 1980s population increase coincided with increases across the species range in stock and rabbit Oryctolagus cuniculus grazing, as well as greater protection and management of sites through protected areas and nature reserves. During the 1990s agri-environment schemes were thought to be the main conservation and policy mechanism driving the favourable conservation status of P. coridon, by facilitating appropriate habitat restoration and management. Weather played a part in the species recovery, with warm, but wet summers associated with increases in abundance. The research provides strong evidence of an important conservation success with the UK Biodiversity Action Plan, with implications for other specialist biodiversity.

PURPOSE: Missense mutations in the splicing factor gene PRPF31 cause a dominant form of retinitis pigmentosa (RP11) with reduced penetrance. Missense mutations in PRPF31 have previously been shown to cause reduced protein solubility, suggesting insufficiency of functional protein as the disease mechanism. Here we examine in further detail the effect of the A216P mutation on splicing function. METHODS: Splicing activity was assayed using an in vivo assay in transfected mammalian cells with rhodopsin (RHO) and transducin (GNAT1) splicing templates. Pull-down assays were used to study the interaction between PRPF31 and one of its cognate partners in the spliceosome, PRPF6. RESULTS: Splicing of RHO intron 3 and GNAT1 introns 3-5 mini-gene templates was inefficient with both spliced and unspliced products clearly detected. Assays using the RHO minigene template revealed a direct negative effect on splicing efficiency of the mutant. However, no effect of the mutation on splicing efficiency could be detected using the longer GNAT1 minigene template or using a full-length RHO transcript, splicing of which had an efficiency of 100%. No unspliced RHO transcripts could be detected in RNA from human retina. Pull-down assays between PRPF31 and PRPF6 proteins showed a stronger interaction for the mutant than wild type, suggesting a mechanism for the negative effect. CONCLUSIONS: Splicing of full-length RHO is more efficient than splicing of the minigene, and assays using a full-length template more accurately mimic splicing in photoreceptors. The RP11 missense mutations exert their pathology mainly via a mechanism based on protein insufficiency due to protein insolubility, but there is also a minor direct negative effect on function.

The Chalkhill Blue Polyommatus coridon is a widespread butterfly of lowland calcareous grassland in southern Britain and is considered a good indicator of habitat condition. Polyommatus coridon has been identified as a Species of Conservation Concern in the UK Biodiversity Action Plan due to a greater than 25% decline in range size since the 1950s, with losses due to the combined effects of habitat destruction, agricultural intensification and neglect. Analysis of annual butterfly monitoring data (transects) collected at 161 sites from 1981 to 2000 show a three-fold population recovery had occurred. The increases were at established sites, with no re-colonisations or range expansion detected. The 1980s population increase coincided with increases across the species range in stock and rabbit Oryctolagus cuniculus grazing, as well as greater protection and management of sites through protected areas and nature reserves. During the 1990s agri-environment schemes were thought to be the main conservation and policy mechanism driving the favourable conservation status of P. coridon, by facilitating appropriate habitat restoration and management. Weather played a part in the species recovery, with warm, but wet summers associated with increases in abundance. The research provides strong evidence of an important conservation success with the UK Biodiversity Action Plan, with implications for other specialist biodiversity.

In Bacillus megaterium, the synthesis of vitamin B-12 (cobalamin) and sirohaem diverges at sirohydrochlorin along the branched modified tetrapyrrole biosynthetic pathway. This key intermediate is made by the action of SirC, a precorrin-2 dehydrogenase that requires NAD(+) as a cofactor. The structure of SirC has now been solved by X-ray crystallography to 2.8 angstrom (1 angstrom = 0.1 nm) resolution. The protein is shown to consist of three domains and has a similar topology to the Multifunctional sirohaem synthases Met8p and the N-terminal region of CysG, both of which catalyse not only the dehydrogenation of precorrin-2 but also the ferrochelation of sirohydrochlorin to give sirohaem. Guided by the structure, in the present study a number of active-site residues within SirC were investigated by site-directed mutagenesis. No active-site general base was identified, although surprisingly some of the resulting protein variants were found to have significantly enhanced catalytic activity. Unexpectedly, SirC was found to bind metal ions such as cobalt and copper, and to bind them in an identical fashion with that observed in Met8p. It is suggested that SirC may have evolved from a Met8p-like protein by loss of its chelatase activity. It is proposed that the ability of SirC to act as a single monofunctional enzyme, in conjunction with ail independent chelatase, may provide greater control over the intermediate at this branchpoint in the synthesis of sirohaem and cobalamin.

Purpose: Missense mutations in the splicing factor gene PRPF31 cause a dominant form of retinitis pigmentosa (RP11) with reduced penetrance. Missense mutations in PRPF31 have previously been shown to cause reduced protein solubility, suggesting insufficiency of functional protein as the disease mechanism. Here we examine in further detail the effect of the A216P mutation on splicing function. Methods: Splicing activity was assayed using an in vivo assay in transfected mammalian cells with rhodopsin (RHO) and transducin (GNAT1) splicing templates. Pull-down assays were used to study the interaction between PRPF31 and one of its cognate partners in the spliceosome, PRPF6. Results: Splicing of RHO intron 3 and GNAT1 introns 3-5 mini-gene templates was inefficient with both spliced and unspliced products clearly detected. Assays using the RHO minigene template revealed a direct negative effect on splicing efficiency of the mutant. However, no effect of the mutation on splicing efficiency could be detected using the longer GNAT1 minigene template or using a full-length RHO transcript, splicing of which had an efficiency of 100%. No unspliced RHO transcripts could be detected in RNA from human retina. Pull-down assays between PRPF31 and PRPF6 proteins showed a stronger interaction for the mutant than wild type, suggesting a mechanism for the negative effect. Conclusions: Splicing of full-length RHO is more efficient than splicing of the minigene, and assays using a full-length template more accurately mimic splicing in photoreceptors. The RP11 missense mutations exert their pathology mainly via a mechanism based on protein insufficiency due to protein insolubility, but there is also a minor direct negative effect on function.

A Lactobacillus reuteri strain isolated from sourdough is known to produce the vitamin cobalamin. The organism requires this for glycerol cofermentation by a cobalamin-dependent enzyme, usually termed glycerol dehydratase, in the synthesis of the antimicrobial substance reuterin. We show that the cobalamin-synthesizing capacity of another L. reuteri strain (20016, the type strain, isolated from the human gut and recently sequenced as F275) is genetically and phenotypically linked, as in the Enterobacteriaceae, to the production of a cobalamin-dependent enzyme which is associated with a bacterial microcompartment (metabolosome) and known as diol dehydratase. We show that this enzyme allows L. reuteri to carry out a disproportionation reaction converting 1,2-propanediol to propionate and propanol. The wide distribution of this operon suggests that it is adapted to horizontal transmission between bacteria. However, there are significant genetic and phenotypic differences between the Lactobacillus background and the Enterobacteriaceae. Electron microscopy reveals that the bacterial microcompartment in L. reuteri occupies a smaller percentage of the cytoplasm than in gram-negative bacteria. DNA sequence data show evidence of a regulatory control mechanism different from that in gram-negative bacteria, with the presence of a catabolite-responsive element (CRE) sequence immediately upstream of the pdu operon encoding diol dehydratase and metabolosome structural genes in L. reuteri. The metabolosome-associated diol dehydratase we describe is the only candidate glycerol dehydratase present on inspection of the L. reuteri F275 genome sequence.

Cells require metal ions as cofactors for the assembly of metalloproteins. Principally one has to distinguish between metal ions that are directly incorporated into their cognate sites on proteins and those metal ions that have to become part of prosthetic groups, cofactors or complexes prior to insertion of theses moieties into target proteins. Molybdenum is only active as part of the molybdenum cofactor, iron can be part of diverse Fe-S clusters or of the heme group, while copper ions are directly delivered to their targets. We will focus in greater detail on molybdenum metabolism because molybdenum metabolism is a good example for demonstrating the role and the network of metals in metabolism: each of the three steps in the pathway of molybdenum cofactor formation depends on a different metal ( iron, copper, molybdenum) and also the enzymes finally harbouring the molybdenum cofactor need additional metal-containing groups to function ( iron sulfur-clusters, heme-iron).

Iron-sulfur proteins are very versatile biological entities for which many new functions are continuously being unravelled. This review focus on their role in the initiation of radical chemistry, with special emphasis on 'radical-SAM' enzymes, since several members of the family catalyse key steps in the biosynthetic pathways of cofactors such as biotin, lipoate, thiamine, heme and the molybdenum cofactor. It will also include other examples to show the chemical logic which is emerging from the presently available data on this family of enzymes. The common step in all the ( quite different) reactions described here is the monoelectronic reductive cleavage of SAM by a reduced [4Fe-4S](1+) cluster, producing methionine and a highly oxidising deoxyadenosyl radical, which can initiate chemically difficult reactions. This set of enzymes, which represent a means to perform oxidation under reductive conditions, are often present in anaerobic organisms. Some other, non-SAM-dependent, radical reactions obeying the same chemical logic are also covered.

Many efforts have been made in recent decades to understand how coenzymes, including vitamins, are synthesised in organisms. In the present review, we describe the most recent findings about the biological roles of five coenzymes: folate (vitamin B9), pantothenate (vitamin B5), cobalamin (vitamin B12), biotin (vitamin B8) and molybdenum cofactor (Moco). In the first part, we will emphasise their biological functions, including the specific roles found in some organisms. In the second part we will present some nutritional aspects and potential strategies to enhance the cofactor contents in organisms of interest.

The evolution of metabolic pathways is discussed with reference to the biosynthesis of a number of vitamins and cofactors. Retrograde and patchwork models are highlighted and their relevance to our knowledge of pathway processes and enzymes is examined. Pathway complexity is explained in terms of the acquisition of broad specificity enzymes

The regulation of cGMP levels is central to the normal process of phototransduction in both cone and rod photoreceptor cells. Two of the proteins involved in this process are the enzyme, retinal guanylate cyclase (retGC), and its activating protein (GCAP) through which activity is regulated via changes in cellular Ca2+ levels. Dominant cone-rod dystrophies arising from changes in retGC1 are essentially restricted to mutations in codon 838 and result in the replacement of a conserved arginine residue with either cysteine, histidine or serine. In all three cases, the effect of the substitution on the in vitro cyclase activity is a loss of Ca2+ sensitivity arising from an increased stability of the coiled-coil domain of the protein dimer and retention of cyclase activity. In contrast, mutations in the Ca2+-coordinating EF hands of GCAP1 result in dominant cone dystrophy; the consequences of these mutations is a reduced ability of the mutant protein to regulate retGC activity in response to changes in Ca2+ levels. Functionally therefore, the retGC2 and GCAP2 mutations are similar in reducing the feedback inhibition of Ca2+ on cyclase activity and thereby on cGMP levels in the photoreceptors.

The regulation of cGMP levels is central to the normal process of phototransduction in both cone and rod photoreceptor cells. Two of the proteins involved in this process are the enzyme, retinal guanylate cyclase (retGC), and its activating protein (GCAP) through which activity is regulated via changes in cellular Ca2+ levels. Dominant cone-rod dystrophies arising from changes in retGC1 are essentially restricted to mutations in codon 838 and result in the replacement of a conserved arginine residue with either cysteine, histidine or serine. In all three cases, the effect of the substitution on the in vitro cyclase activity is a loss of Ca2+ sensitivity arising from an increased stability of the coiled-coil domain of the protein dimer and retention of cyclase activity. In contrast, mutations in the Ca2+-coordinating EF hands of GCAP1 result in dominant cone dystrophy; the consequences of these mutations is a reduced ability of the mutant protein to regulate retGC activity in response to changes in Ca2+ levels. Functionally therefore, the retGC2 and GCAP2 mutations are similar in reducing the feedback inhibition of Ca2+ on cyclase activity and thereby on cGMP levels in the photoreceptors.

This study investigates the functional consequences of two mutations, A194E and A216P, in the splicing factor gene PRPF31 linked to autosomal dominant retinitis pigmentosa (RP11). Using a yeast complementation assay, we demonstrate that introduction of the human A216P mutation into the yeast orthologue PRP31p results in only partial rescue of growth at the restrictive temperature, indicating that splicing function is not fully restored. An in vivo assay of splicing function in human cells using a bovine rod opsin splicing template did not detect any defect in splicing efficiency or accuracy attributable to either mutation, suggesting that neither has a dominant negative effect on splicing. However, western analysis and immunofluorescence microscopy of mammalian cells transfected with PRPF31 revealed that both mutations substantially hinder translocation of the protein into the nucleus. The overall effect may thus be an insufficiency in splicing function, which is revealed only under conditions of elevated splicing demand. With the need to replenish disc proteins on a daily basis, such conditions will exist in rod photoreceptors and this may underlie the disease pathology

Two enzymes which play an important role in regulation and flux control through the tetapyrrole biosynthetic pathway are considered. The Rhodobacter sphaeroides 5-aminolaevulinic acid synthase isoenzymes are discussed and the progress being made on their recombinant expression and isolation is reported. The Escherichia coli uroporphyrinogen methylase, which is encoded by the cysG gene, is also examined. In this case evidence is provided which demonstrates that the gene product is responsible for the complete synthesis of sirohaem from uroporphyrinogen III. The enzyme is thus capable of performing two S-adenosylmethionine-dependent methylation reactions, an NADP(+)-dependent dehydrogenation and iron chelation. The uroporphyrinogen methylase is thus a small multifunctional enzyme.

Pp.3-32p ; 4⁰., Dated on p.29: Bury, Feb. 27, 1732-33., Title from opening words., 'Advertisement' at end requests information for "Dr. Warren, at Bury St. Edmund's", who may be Martin Warren., Reproduction of original from the British Library., English Short Title Catalog, ESTCT10217., Electronic data. Farmington Hills, Mich. : Thomson Gale, 2003. Page image (PNG). Digitized image of the microfilm version produced in Woodbridge, CT by Research Publications, 1982-2002 (later known as Primary Source Microfilm, an imprint of the Gale Group)., (DLPS) 004789409.0001.000, (ESTC) T10217, http://quod.lib.umich.edu/t/text/accesspolicy.html, To the extent possible under law, the Text Creation Partnership has waived all copyright and related or neighboring rights to this keyboarded and encoded edition of the work described above, according to the terms of the CC0 1.0 Public Domain Dedication (http://creativecommons.org/publicdomain/zero/1.0/). This waiver does not extend to any page images or other supplementary files associated with this work, which may be protected by copyright or other license restrictions. Please go to http://www.lib.umich.edu/tcp/ecco/ for more information.